Modified viruses

ABSTRACT

This document relates to methods and materials for making and using viruses (e.g., measles viruses or adenoviruses) having a reduced susceptibility to antibody neutralization (e.g., antibody neutralization by serum from measles virus vaccines). For example, recombinant morbilliviruses (e.g., recombinant measles viruses) having a modified H gene and a modified F gene, as well as methods of using a recombinant virus are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/612,842, filed Nov. 12, 2019, which is a National Stage applicationunder U.S.C. § 371 of International Application No. PCT/US2018/026079,filed Apr. 4, 2018, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/506,892, filed on May 16, 2017. The disclosuresof the prior applications are considered part of (and are incorporatedby reference in) the disclosure of this application.

BACKGROUND 1. Technical Field

This document relates to methods and materials for making and usingviruses (e.g., measles viruses or adenoviruses) having a reducedsusceptibility to antibody neutralization (e.g., antibody neutralizationby serum from measles virus vaccines).

2. Background Information

Measles virus (MV) causes many deaths each year, mostly amongst childrenunder the age of five. Unvaccinated children are at highest risk ofmeasles and measles related deaths. In particular, infants whosematernal anti-measles antibody titers have waned to non-protectivelevels, but are still too young to receive the current measles vaccinerecommended for infants at 9-12 months, can be at an elevated risk ofmeasles and measles related deaths.

In addition, Edmonston lineage MVs have proven to be potentially potentanticancer drugs when administered to measles seronegative cancerpatients. However, upwards of 90% of cancer patients in most countrieshave protective titers of anti-measles antibodies in their blood (e.g.,due to natural measles infection or measles vaccination). Measles-immunehuman serum negates the therapeutic efficacy of systemicallyadministered oncolytic MVs in tumor-bearing mice by neutralizing thevirus before it reaches its target (the tumor cells). Hence, themajority of cancer patients are unable to benefit from systemicallyadministered oncolytic MV therapy.

SUMMARY

This document provides methods and materials for making and usingviruses (e.g., MVs) having a reduced susceptibility to antibodyneutralization (e.g., antibody neutralization by monoclonal anti-MVantibodies and/or serum from MV vaccines). For example, this documentprovides recombinant morbilliviruses (e.g., recombinant MVs) having areduced ability of being recognized by anti-MV antibodies that weregenerated against a wild-type MV or a pre-existing MV vaccine ascompared to a wild-type MV H and F polypeptides or the H and Fpolypeptides of a pre-existing MV vaccine.

As demonstrated herein, a recombinant MV with a substituted H gene(e.g., encoding a multiply mutated measles H protein from which severalimmunodominant epitopes have been eliminated) and a substituted F gene(e.g., a canine distemper virus F gene encoding a canine distemper virusF protein) is resistant to neutralization by human measles-immune humanserum. A recombinant MV described herein can be used to address themajor limitations of currently available measles vaccines, MV-basedplatform vaccines, and oncolytic MVs, namely their susceptibility toneutralization by measles-immune human serum.

In one aspect, this document features a virus having a nucleic acidencoding a measles virus H polypeptide comprising at least 6 amino acidsubstitutions and a nucleic acid encoding a morbillivirus F polypeptideother than a measles virus F polypeptide. The virus can be a measlesvirus. The virus can be a viral vector (e.g., vector derived from anadenovirus, an adeno-associated virus, a retrovirus, a lentivirus, aherpes virus, a vaccinia virus, or a rhabdovirus). The measles virus Hpolypeptide can include SEQ ID NO:9 having 6 or more (e.g., 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following amino acidsubstitutions: S189P, E235G N238D, L249P, G302R, Y310C, Q311R, R377Q,M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G andF552V. In some cases, the measles virus H polypeptide can include SEQ IDNO:9 having a E471K substitution. For example, the measles virus Hpolypeptide can include SEQ ID NO:9 having the following amino acidsubstitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q,M378K, D416N, E471K, N481Y, K488E, G491E, H495R, D505T, R533G, S546G,R547G and F552V. In some cases, the measles virus H polypeptide caninclude SEQ ID NO:1 having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, or 36) of the following amino acid substitutions: H175,D149N, S189P, G211S, E235G, N238D, S240N, L249P, V280I, N282K, G302R,E303G, Q311R, Q334H, A359T, K364N, R377Q, M378K, P397L, T420A, V421A,L423P, F476L, N481Y, G491D, H495R, D505T, R533G, V562T, D574A, K576R,I594L, G603E, T609N, G613E, and T614A. For example, the said measlesvirus H polypeptide can include SEQ ID NO:3. In some cases, the measlesvirus H polypeptide can include SEQ ID NO:1 having a E471K substitution.For example, the measles virus H polypeptide can include SEQ ID NO:1having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or36) of the following amino acid substitutions: H17S, D149N, S189P,G211S, E235G, N238D, S240N, L249P, V280I, N282K, G302R, E303G, Q311R,Q334H, A359T, K364N, R377Q, M378K, P397L, T420A, V421A, L423P, E471K,F476L, N481Y, G491D, H495R, D505T, R533G V562T, D574A, K576R, I594L,G603E, T609N, G613E, and T614A. The measles virus F polypeptide can be acanine distemper virus F polypeptide. The virus can exhibitCD46-dependent cell entry. The virus can exhibit reduced (or no)Nectin-4-dependent cell entry relative to a wild type virus. The viruscan lack the measles virus F polypeptide, can lack nucleic acid encodingthe measles virus F polypeptide, or can lack both the measles virus Fpolypeptide and the nucleic acid encoding the measles virus Fpolypeptide. The virus can lack the wild-type measles virus Hpolypeptide, can lack nucleic acid encoding the wild-type measles virusH polypeptide, or can lack both the wild-type measles virus Hpolypeptide and the nucleic acid encoding the wild-type measles virus Hpolypeptide.

In another aspect, this document features a method for reducing thenumber of viable tumor cells in a mammal. The method includes, orconsists essentially of, administering to the mammal a virus having anucleic acid encoding a measles virus H polypeptide comprising at least17 amino acid substitutions and a nucleic acid encoding a morbillivirusF polypeptide other than a measles virus F polypeptide. The virus can bea measles virus. The virus can be viral vector (e.g., vector derivedfrom an adenovirus, an adeno-associated virus, a retrovirus, alentivirus, a herpes virus, a vaccinia virus, or a rhabdovirus). Themeasles virus H polypeptide can include SEQ ID NO:9 having 6 or more(e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of thefollowing amino acid substitutions: S189P, E235G, N238D, L249P, G302R,Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T,R533G, S546G, R547G, and F552V. In some cases, the measles virus Hpolypeptide can include SEQ ID NO:9 having a E471K substitution. Forexample, the measles virus H polypeptide can include SEQ ID NO:9 havingthe following amino acid substitutions: S189P, E235G, N238D, L249P,G302R, Y310C, Q311R, R377Q, M378K, D416N, E471K, N481Y, K488E, G491E,H495R, D505T, R533G, S546G, R547G, and F552V. In some cases, the measlesvirus H polypeptide can include SEQ ID NO:1 having 6 or more (e.g., 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) of the following aminoacid substitutions: H175, D149N, S189P, G2115, E235G, N238D, 5240N,L249P, V280I, N282K, G302R, E303G, Q311R, Q334H, A359T, K364N, R377Q,M378K, P397L, T420A, V421A, L423P, F476L, N481Y, G491D, H495R, D505T,R533G, V562T, D574A, K576R, I594L, G603E, T609N, G613E, and T614A. Forexample, the said measles virus H polypeptide can include SEQ ID NO:3.In some cases, the measles virus H polypeptide can include SEQ ID NO:1having a E471K substitution. For example, the measles virus Hpolypeptide can include SEQ ID NO:1 having 6 or more (e.g., 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36) of the following amino acidsubstitutions: H17S, D149N, S189P, G211S, E235G, N238D, 5240N, L249P,V2801, N282K, G302R, E303G, Q311R, Q334H, A359T, K364N, R377Q, M378K,P397L, T420A, V421A, L423P, E471K, F476L, N481Y, G491D, H495R, D505T,R533G, V562T, D574A, K576R, I594L, G603E, T609N, G613E, and T614A. Themeasles virus F polypeptide can be a canine distemper virus Fpolypeptide. The virus can exhibit CD46-dependent cell entry. The viruscan exhibit reduced (or no) Nectin-4-dependent cell entry relative to awild type virus. The mammal can be a human.

In another aspect, this document features a method for stimulating animmune response against measles virus in a mammal. The method includes,or consists essentially of, administering to the mammal a virus having anucleic acid encoding a measles virus H polypeptide comprising at least17 amino acid substitutions and a nucleic acid encoding a morbillivirusF polypeptide other than a measles virus F polypeptide. The virus can bea measles virus. The virus can be viral vector (e.g., vector derivedfrom an adenovirus, an adeno-associated virus, a retrovirus, alentivirus, a herpes virus, a vaccinia virus, or a rhabdovirus). Themeasles virus H polypeptide can include SEQ ID NO:9 having 6 or more(e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of thefollowing amino acid substitutions: S189P, E235G N238D, L249P, G302R,Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T,R533G, S546G, R547G and F552V. In some cases, the measles virus Hpolypeptide can include SEQ ID NO:9 having a E471K substitution. Forexample, the measles virus H polypeptide can include SEQ ID NO:9 havingthe following amino acid substitutions: S189P, E235G, N238D, L249P,G302R, Y310C, Q311R, R377Q, M378K, D416N, E471K, N481Y, K488E, G491E,H495R, D505T, R533G, S546G, R547G, and F552V. In some cases, the measlesvirus H polypeptide can include SEQ ID NO:1 having 6 or more (e.g., 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) of the following aminoacid substitutions: H17S, D149N, S189P, G211S, E235G, N238D, S240N,L249P, V280I, N282K, G302R, E303G, Q311R, Q334H, A359T, K364N, R377Q,M378K, P397L, T420A, V421A, L423P, F476L, N481Y, G491D, H495R, D505T,R533G, V562T, D574A, K576R, I594L, G603E, T609N, G613E, and T614A. Forexample, the said measles virus H polypeptide can include SEQ ID NO:3.In some cases, the measles virus H polypeptide can include SEQ ID NO:1having a E471K substitution. For example, the measles virus Hpolypeptide can include SEQ ID NO:1 having 6 or more (e.g., 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36) of the following amino acidsubstitutions: H17S,

D149N, S189P, G2115, E235G N238D, 5240N, L249P, V280I, N282K, G302R,E303G Q311R, Q334H, A359T, K364N, R377Q, M378K, P397L, T420A, V421A,L423P, E471K, F476L, N481Y, G491D, H495R, D505T, R533G, V562T, D574A,K576R, I594L, G603E, T609N, G613E, and T614A. The measles virus Fpolypeptide can be a canine distemper virus F polypeptide. The virus canexhibit CD46-dependent cell entry. The virus can exhibit reduced (or no)Nectin-4-dependent cell entry relative to a wild type virus. The mammalcan be an infant (e.g., a human infant). The human infant can havetransplacentally acquired anti-measles antibodies.

In another aspect, this document features a nucleic acid constructcomprising (or consisting essentially of, or consisting of) a nucleicacid encoding a measles virus H polypeptide comprising at least sixamino acid substitutions as compared to a wild-type measles virus Hpolypeptide, and a nucleic acid encoding a morbillivirus F polypeptideother than a measles virus F polypeptide. The measles virus Hpolypeptide can comprise SEQ ID NO:9 having six or more of the followingamino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C,Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G,S546G, R547G, and F552V. The measles virus H polypeptide can compriseSEQ ID NO:9 having the following amino acid substitutions: S189P, E235GN238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E,G491E, H495R, D505T, R533G, S546G, R547G, and F552V. The measles virus Hpolypeptide can comprise at least one amino acid substitution withineach of the antigenic sites set forth in Table 1. The wild-type measlesvirus H polypeptide can be a wild-type measles virus H polypeptide ofthe MVi/Madrid.SPA/50.10 strain. The wild-type measles virus Hpolypeptide can comprise the amino acid sequence set forth in SEQ IDNO:9. The measles virus H polypeptide can comprise SEQ ID NO:3. Themorbillivirus F polypeptide can be a canine distemper virus Fpolypeptide. The nucleic acid construct can be a viral vector. The viralvector can be derived from a virus selected from the group consisting ofan adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, aherpes virus, a vaccinia virus, and a rhabdovirus. The measles virus Hpolypeptide can comprise at least one amino acid substitution withineach of the antigenic sites set forth in Table 2. The measles virus Hpolypeptide can comprise a substitution at position E471 as compared tothe wild-type measles virus H polypeptide. The substitution at positionE471 can be a E471K substitution. The measles virus H polypeptide cancomprise SEQ ID NO:9 having the following amino acid substitutions:S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N,E471K, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, andF552V. The nucleic acid construct can be a construct that does notencode the measles virus F polypeptide. The nucleic acid construct canbe a construct that does not encode the wild-type measles virus Hpolypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 presents mutations encoded in the MV-H polypeptide to escapeneutralization.

FIG. 2 shows that mutations in MV-H circumvent neutralizing antibodies,but F-specific antibodies neutralize MV.

FIG. 3 includes schematics of MV, CDV, and MVs encoding either CDV H,CDV F, or both, and their correspondent neutralization sensitivity bywhole pooled human sera or MV H/F specifically absorbed.

FIG. 4 depicts the tropism and fusion capacity for a mutated MV Hpolypeptide in conjunction with a CDV F polypeptide.

FIG. 5 illustrates that a multiple mutated MV H polypeptide can inducebroadly neutralizing antibodies against multiple MV strains.

FIGS. 6A-6I contain exemplary sequences of MV polypeptides and nucleicacids encoding MV polypeptides. FIG. 6A shows a naturally occurring MV Hpolypeptide (SEQ ID NO:1). FIG. 6B shows a nucleic acid encoding anaturally occurring MV H polypeptide (SEQ ID NO:2). FIG. 6C shows amodified H polypeptide (SEQ ID NO:3). FIG. 6D shows a naturallyoccurring MV F polypeptide (SEQ ID NO:4). FIG. 6E shows a nucleic acidencoding a naturally occurring MV F polypeptide (SEQ ID NO:5). FIGS. 6Fand 6G show CDV F polypeptides (SEQ ID NOs: 6 and 7). FIG. 6H shows aCDV H polypeptide (SEQ ID NO:8). FIG. 61 shows and a naturally occurringMV H polypeptide of MVi/Madrid.SPA/50.10[H1] (SEQ ID NO:9).

FIGS. 7A-E. Reverse genetic system for Stealth virus rescue:Optimization of antigenome-expressing plasmid. (FIG. 7A) Number of cellsexpressing GFP as determined by flow cytometry three days aftertransfection of the BHK rescue cells. (FIG. 7B) Virus production on daythree after transfection was determined by freeze-thaw rescue cells (P₀)and titrating on Vero cells. (FIG. 7C) Scatter plot showing thecorrelation between rescue efficiency and virus production. (Pearsonr=0.789, p<0.05). Dashes lines represent 95% confidence interval. (FIG.7D) Rescue of recombinant viruses. Rescue cells were overlaid 3 daysafter transfection onto Vero cells, and syncytia formation was evaluated3 days afterwards. * indicates lack of representativeness in themicrophotograph. Arrow indicates a single GPF-positive cells. After fivesemi-blind passages, Stealth virus started to spread similarly to theparental virus. (FIG. 7E) Plasmid constructs used in this study. Shownare the key sequence variations between constructs. T_(min) is a minimalT7 promoter and the addition of GGGAGA (SEQ ID NO:10) drives higherlevels of gene expression (T_(opt)). However, an optimal T7 promoterrequires a self-cleaving hammerhead ribozyme (HHrbz) (Yun et al., J.Virol., 89(2):1242-53 (2015)). Construct 1 (SEQ ID NO:11) and Construct2 (SEQ ID NO:12) are shown. Construct 3 (SEQ ID NO:13) was describedelsewhere (Beaty et al., mSphere, 2(2):ppi00376-16 (2017)). Construct 4(SEQ ID NO:14) has additionally a 5′ the Elongation Factor-1α corepromoter (hEFL-HTLV) followed by a chimeric intron sequence. Allconstructs had the Hepatitis delta ribozyme (HDV) and T7 terminatordownstream of the MeV antigenome.

FIGS. 8A-B. Engraftment of CD46 footprint onto MeV-H genotype H1. (FIG.8A) Left panel. CHO cells expressing or not the cellular receptors(SLAM, CD46 and nectin-4) were infected with GFP-expressing MeV encodingthe indicated MeV-H, at moi 0.1. eGFP autofluorescence was recorded 72hours post infection. (FIG. 8A) Right panel. Cell-fusion triggered byco-expression of vaccine MeV-F and the indicated MeV-H. Cells werestained 48 hours later with Giemsa and microphotograph were taken.Magnification, x40. (FIG. 8B) Kinetics of cell-cell fusion as determinedby split luciferase assay.

FIGS. 9A-B. Antigenic variation in 47 viruses. (FIG. 9A) Model of thedimeric structure of MeV-H47 with N-linked sugar attached. N-linkedsugars are depicted as black (N168-, N200-, N215linked) or orangespheres (N416-linked). Amino acid differences with regard to the MeV-Hvaccine Moraten strain are indicated. Blue, MeV-H-genotype-H1-specificchanges; Red, engineered nAb-escape mutations. (FIG. 9B) Antigenic mapof viruses encoding different MeV-H proteins. Antigenicity wasdetermined by virus plaque reduction microneutralization (PRMN) assayand color-coded according to the Log₂ NT50 differences. Individualneutralization curves are shown in FIG. 19.

FIGS. 10A-D. BH030 defines a novel antigenic site onto MeV-H. (FIG. 10A)PRMN assay of mAb BH030 against recombinant MeV encoding differentgenotype-specific MeV-H. Data points were fitted by non-linearregression analysis with Graph Pad Prism. Inhibitory concentration 50%(IC₅₀) is indicated by a dotted line. Of note, C2 viruses selectivelyescape neutralization. (FIG. 10B) Amino acid sequence alignment for theputative BH030 epitope, showing the differential amino acid substitutionin C2 viruses. Consensus is set forth as SEQ ID NO:15. (FIG. 10C) H1viruses show resistance against BH030 neutralization in comparison withA viruses and A mutant (416DLS→NLS), but are still neutralized, as Δ7viruses are. The addition of E471E into Δ7, name herein Δ8, results inBH030 escape. (FIG. 10D) Putative antigenic site defined by mnAb BH030.Glu471 is not masked by N416-linked sugar, which shields site IIb, andtherefore define a putative new antigenic site, V, that expands fromsites IIb to site III.

FIGS. 11A-C. Immunogenicity of a drifted MeV-H. (FIG. 11A) Schematicrepresentation of the experiment. C57BL6 mice were intravenouslyinjected within 5 sec with expression plasmids for GFP (negativecontrol) or different MeV-H variants. One month later, neutralizingantibodies activity was assessed by PRMN assay against vaccine virus(FIG. 11B) or Δ8 virus (FIG. 11C). Each data point represents a singleindividual. Missing points were due to animal death or scarcity ofmaterial. Data from two different immunizations are included. Greyshading indicates a level of neutralizing antibodies consideredunprotective against the disease. Lowe dotted lines indicate the limitof detection for the assay.

FIGS. 12A-D. Δ8 viruses escape a broader anti-MeV-H polyclonal response.(FIG. 12A) Left panel, PRMN assay of rabbit anti-MeV-H polyclonalantibodies against vaccine and MeV-H mutant viruses (Δ7 and Δ8). Curveswere fitted by nonlinear regression to calculate NT₅₀ values. Rightpanel. Neutralization titer differences (ND) between viruses. The samerabbit anti-MeV-H polyclonal antibodies were further tested against apanel of recombinant viruses encoding genotype-specific MeV-H genes orthe Δ8 mutant, the NT₅₀ was calculated as in the left panel, and the NDare plot as log₂. A difference ≥2 log₂ is considered substantiallyantigenic different (dotted line). (FIG. 12B) Monitoring of MeVglycoprotein depletion conditions. IgG antibody levels after human seraincubation with cells non-expressing or expressing either of MeVglycoproteins MeV-H and MeV-F (FIG. 20), compared to those levels inuntreated human sera (condition 0). Serum samples were diluted 1:10 inculture medium and subsequently culture for 4 days onto a monolayer ofMel-JuSo cells (Mel-JuSo/wt, condition 1; Mel-Ju-So-H, condition 2;Mel-JuSo-F, condition 3). Supernatants were collected and tested for thepresence of MeV-F and MeV-H-specific antibodies by a FACS-measuredimmunofluorescence assay. Dotted line encloses values considerednegative. (FIG. 12C) VCA IgG levels in U/mL remaining afterMeV-F-specific depletion condition. (FIG. 12D) Neutralization capacityof poyclonal human sera against vaccine and Δ8 viruses. Sera sampleswere collected from healthy vaccinated young adults and were leftuntreated or depleted from MeV-F-specific antibodies. Epstein-Barr virus(EBV) VCA IgG levels were used to make comparison of the neutralizationpotency between conditions, accounting for dilution factors betweenthem.

Data are presented as the mean of individual sera samples (N=6). Trendline of neutralization curve across samples was determined by non-linearregression fitting. ***P<0.0005, Wilcoxon matched-pairs signed ranktest.

FIGS. 13A-B. Role of MeV-H and MeV-H in virus neutralization. (FIG. 13A)MeV glycoprotein specificity of pooled human sera. Conditions and IgGspecific levels were described and determined as in FIG. 12. Data areshown as histogram plots. (FIG. 13B) PRNM of envelope-exchange viruses.Isogenic rMeV encoding MeV envelope glycoproteins (virus 1, denoted inred) or CDV (virus 2, denoted in blue) as well as viral chimera betweenthem (virus 3 and 4), were used for the neutralization sensitivityagainst human sera preicubated with control cells (non absorbed) orcells expressing either MeV-H or MeV-F glycoproteins. Representativesyncitia are shown.

FIGS. 14A-D. Characterization of recombinant MeVs. (FIG. 14A)Replication kinetics of recombinant viruses. Vero/hSLAM were infectedwith the indicated virus at MOI of 0.03. At the indicated time, GFPautofluorescence and bright-field images were recorded and overlayed.Magnification, x40. Afterwards, cells were collected in the culturemedium, and virus titer was determined on Vero/hSLAM as FFU/mL. A, H1,and Δ8 denotes MeV expressing the equivalent MeV-H genes and vaccineMeV-F, whilst Stealth virus is a virus encoding MeV-HΔ8 together withCDV-F. (FIG. 14B) Protein incorporation into virions. 10⁴ virusparticles were electrophoresed under reducing conditions andimmunoblotted with the relevant antibody. Note that anti-MeV-F antibodydoes not cross-react with CDV-F. (FIG. 14C) Left panel. PRMN NT₅₀ valuesof measles immune human sera against the MeV A (vaccine) and Stealthviruses. Each line represents a single individual (N=14). Dotted lineindicated the antibody levels threshold for protection against clinicaldisease. Ferret serum anti-CDV was used as a control. ***, P<0.001 asdetermined by Wilcoxon matched-pairs signed rank test. Right panel,correlation between NT₅₀ for the vaccine virus and Stealth. NT₅₀ valuesare plotted on a log₂ scale. P<0.001, in both Pearson and Spearmancorrelation test. Dotted lines indicated the 95% confidence interval forthe regression analysis. R=0.51. (FIG. 14D) PRMN of guinea Pigpolyclonal MeV antiserum against A and Stealth viruses. Non-linearregression was performed to calculate the NT₅₀ values, which weretransformed to mIU/mL (3584 for A virus and 563 for Stealth).

FIGS. 15A-D. Comparison of the receptors' footprint in MeV-H. (FIG. 15A)Schematic representation of the MeV-H primary sequence. From left toright: C, cytoplasmic tail; T, transmembrane domain; Stalk domain;(β1-6, beta-propeller blades 1-6. Amino acid positions delineating thedifferent domains are indicated. (FIG. 15B to FIG. 15D) MeV-H is shownas a ribbon with a rainbow coloring consistent with FIG. 15A, whereasthe receptors are shown as translucent surface colored in cyan (SLAM,FIG. 15B), magenta (nectin-4, FIG. 15C) and blue (CD46, FIG. 15D).Spheres denote residues ≤4.5 Å from receptor entity SLAM (PDB: 3ALZ),nectin-4 (PDB: 4GJT) and CD46 (PDB: 3INB), colored in cyan, magenta andblue, respectively. In each MeV-H-receptor complex structure, spheresare colored differently to the receptor coloring if the residue is alsorelevant for the interaction with in any of the others receptors, andcolored accordingly. Residue Y524 relevant for the interaction with allthree receptors is colored in orange. For comparison, the MeV-Horientation is kept consistent across panels. Stick representationdepict residues structurally involved as defined by ≤4.5 Å from thereceptor entity, but not functionally (L464, L482, F483, L500, D530,Y543, S548) and vice versa (V451, N-Y481, K488, P497) (Mateo et al., J.Virol., 87(16):9208-16 (2013)).

FIGS. 16A-D. Receptor usage for Stealth. (FIG. 16A) Stealth virusinfection of a panel of CHO cells expressing or not the MeV receptors.Infection was done at MOI 0.03 and recorded 3 days later. Magnificationx40. (FIG. 16B) Flow cytometry to determine the surface expression ofthe MeV receptors in the CHO panel. Cells were stained with the relevantantibody, and the number of molecules per cell was determined withQuantibrite BD. (FIG. 16C) Kinetics fusion assay after co-expression ofMeV-F vaccine strain with either MeV-H vaccine strain or MeV-M.8. Mean±standard deviation (n=3). (FIG. 16D) Binding of MeV receptors-Fc toplastic-bound MeV-H protein, monitored by OD (FIG. 18). Data points arepresented as mean ±standard deviation (n=3) and were fitted to aone-site mode of total binding (R²≥0.99). *P<0.05***P<0.005;***P<0.0001.One-way ANOVA with the Bonferroni's multiple comparisons.

FIG. 17. Kinetic fusion assay on vero cells. MeV-H vaccine strain or 48were co-transfected together with MeV-F vaccine strain. Fusion wasquantified by split-luciferase assay. Transfection of MeV-F alone servedas negative control. Mean ±standard deviation of a representativeexperiment performed in triplicate.

FIG. 18. ELISA method for determination of receptor binding to MeV-H.Precoated microwells with Strep-tactin XT were incubated withsupernatant containing MeV-H, followed by incubation with eitheranti-FLAG antibody (control) or single receptor-Fc. Binding was revealedby HRP-conjugated anti-IgG and monitored by optical density.

FIG. 19. PRMN assay of anti-MeV-H mAbs. Results are expressed as % ofvirus control in the absence of mAb. Data are presented as mean±standard deviation of two independent experiments performed inquadruplicate.

FIG. 20. Flow cytometry to characterize MeV-glycoprotein expression onMel-JuSo cell lines. Parental Mel-JuSO cells and Mel-JuSO cellsexpressing either MeV-H or MeV-F were stained with anti-MeV-H (C28-10-8)or anti-MeV-F (F3-5) mAb and results are shown as histograms.

FIG. 21. Correlation analysis for levels of IgG MeV-H and MeV-F-specificantibodies. Black dots represent single vaccinated individuals. Arrowindicates a pooled human sera (Valley Biomedical). Statistical analysisperformed with a two-tailed Pearson correlation.

DETAILED DESCRIPTION

This document provides nucleic acids, polypeptides, and virusescontaining the nucleic acids and/or polypeptides. This document alsoprovides methods for using the viruses to treat cancer patients or tovaccinate infants to help protect them from MV infections. For example,this document provides MV hemagglutinin (H) polypeptides, nucleic acidsencoding MV H polypeptides, CDV F polypeptides, nucleic acid sequencesencoding CDV F polypeptides, and viruses containing such nucleic acidsand/or polypeptides. For example, this document provides a recombinantvirus (e.g., a MV or an adenovirus (Ad)) containing a nucleic acidencoding a modified H polypeptide and a nucleic acid encoding a modifiedF polypeptide. Such a recombinant virus can exhibit reducedsusceptibility to antibody neutralization, reduced ability to triggermembrane fusion, and/or diminished replicative fitness. A recombinantvirus described herein can propagate in cells (e.g., human cells such asVero cells and HeLa cells) as efficiently as a wild type virus. Theviruses described herein can be used to treat cancer patients or tovaccinate infants in a manner such that the viruses exhibit a reducedsusceptibility to antibody neutralization. In some cases, a recombinantvirus described herein can be used to treat cancer in patients who havepre-existing measles immunity. In some cases, a recombinant virusdescribed herein can be used to vaccinate children having neutralizinganti-measles antibodies (e.g., transplacentally acquired neutralizinganti-measles antibodies).

This document provides H polypeptides, F polypeptides, and nucleic acidsthat encode them that are heterologous to naturally occurring H and Fpolypeptides.

The term “nucleic acid” as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. A nucleic acid can be double-stranded orsingle-stranded. A single-stranded nucleic acid can be the sense strandor the antisense strand. In addition, a nucleic acid can be circular orlinear.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acids that are present in a viral genome, includingnucleic acids that normally flank one or both sides of the nucleic acidin a viral genome. The term “isolated” as used herein with respect tonucleic acids also includes any non-naturally-occurring nucleic acidsequence, since such non-naturally-occurring sequences are not found innature and do not have immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a nucleic acidconstruct (e.g., a vector such as an expression vector, an autonomouslyreplicating plasmid, or a virus (e.g., a paramyxovirus, retrovirus,lentivirus, Ad, herpes virus, adenovirus, parvovirus such as anAd-associated virus, rhabdovirus such as vesicular stomatitis virus, orvaccinia virus), or into the genomic DNA of a prokaryote or eukaryote.In cases where an isolated nucleic acid is a virus, the virus can be,for example, an oncolytic virus or a viral vector (e.g., a viral genetransfer vector). For example, a viral vector can be a vector derivedfrom an Ad, an Ad-associated virus, a retrovirus, a lentivirus, a herpesvirus, a vaccinia virus, or a rhabdovirus. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not considered an isolated nucleicacid.

As used here, a “polypeptide” refers to a chain of amino acid residues,regardless of post-translational modification (e.g., phosphorylation orglycosylation).

A nucleic acid encoding a modified H polypeptide provided herein canencode a MV H polypeptide that is heterologous to naturally occurring MVH polypeptides or to the H polypeptide having the amino acid sequenceset forth in GenBank Accession No. AAF85673 (Version AAF85673.1, GI No.9181880; SEQ ID NO:1). Additional examples of naturally occurring MV Hpolypeptides (and the nucleic acid sequences encoding them) can be foundin public databases. For example, GenBank Accession No. KP191044(Version KP191044.1, GI No. 727347518; SEQ ID NO:2) provides an exampleof a nucleic acid sequence encoding a wild type H polypeptide. In somecases, a MV H polypeptide designed to be heterologous to naturallyoccurring MV H polypeptides and/or heterologous to the H polypeptidehaving the amino acid sequence set forth in SEQ ID NO:1 can be referredto as a modified H polypeptide. The term “H polypeptide amino acidsequence” as used herein refers to an amino acid sequence that is atleast 85 percent (e.g., at least 85, 90, 95, 99, or 100 percent)identical to the sequence set forth in SEQ ID NO:1. In some cases, amodified H polypeptide can have at least 6 (e.g., 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) aminoacid residues associated with immunodominant epitopes of the MV Hglycoprotein that have been modified (e.g., substituted). Amino acidsubstitutions in H polypeptides typically are located at positionsinvolved in the binding of H polypeptides to its receptors.

In some cases, a modified H polypeptide can have one or more amino acidsubstitutions in, for example, each of the 6 antigenic sites set forthin Table 1.

TABLE 1 Antigenic sites of H polypeptides. Site Name Start Position StopPosition 1 Ia, V, VI, E4, 1, 307 318 LE 2 Ib, NE, IV 233 250 3 IIa, SSE,VII, 2 487 495 4 IIb, SSE, VII, 2 471 477 5 III, VII, E2, IIIB, 530 5623, 4, RBE 6 ‘Noose’, HNE, I, 377 405 E3, D, D/E

In some cases, a modified H polypeptide can have one or more amino acidsubstitutions in, for example, 7 of the 8 antigenic sites set forth inTable 2 or each of the 8 antigenic sites set forth in Table 2.

TABLE 2 Antigenic sites of H polypeptides. Site Name Start Position StopPosition 1 E1, ϕ 280 285 2 Ia, V, VI, E4, 1, 307 318 LE 3 Ib, NE, IV 233250 4 IIa, SSE, VII, 2 487 495 5 IIb, SSE, VII, 2 471 477 6 III, IIIA,VII, E2, 530 562 IIIB, 3, 4, RBE 7 ‘Noose’, HNE, I, 377 405 E3, D, D/E 8V 450 456 464 470 478 484

In some cases, a modified H polypeptide can have 6 or more (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore) amino acid substitutions in, for example, positions correspondingto amino acids D149, A158, T174, T176, T177, F180, L181, S189, R195,N200, R211, R212, V220, E235, S240, G243, L246, L249, H252, V259, F276,V280, D283, S285, L296, G302, E303, S305, P308, Q311, S316, S318, M333,Q334, P338, L339, V345, I346, L351, V357, A359, K364, V367, R377, M378,F382, A392, C394, P397, V412, T420, V421, L423, K424, H448, V450, K460,E471, I473, F476, K477, N481, G491, E/G492, H495, D505, L517, R533,I559, V562, I564, D574, Q575, K576, A587, G603, V608, T609, E611, G613,T614, R616, and/or R617 of a full-length H polypeptide having the aminoacid sequence set forth in SEQ ID NO:1. For example, a modified Hpolypeptide provided herein can have the amino acid sequence set forthin SEQ ID NO:1 with the exception that the MV H polypeptide has 6 ormore (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, or more) of the following amino acid substitutions:H175, D149N, S189P, G2115, E235G

N238D, S240N, L249P, L276G, V280I, G302R, E303G, Q311R, Q334H, A359T,K364N, R377Q, M378K, P397L, T420A, V421A, L423P, E471K, F476L, N481Y,G491D, H495R, D505T, R533G, I594L, V562T, D574A, K576R, G603E, T609N,G613E, and T614A.

In some cases, a modified H polypeptide can have 6 or more (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acidsubstitutions in, for example, positions corresponding to amino acidsS189, E235, N238, L249, G302, Y310, Q311, R377, M378, D416, E471, N481,K488, G491, H495, D505, R533, S546, R547, and/or F552 of a full-length Hpolypeptide having the amino acid sequence set forth in SEQ ID NO:9. Forexample, a modified H polypeptide provided herein can have the aminoacid sequence set forth in SEQ ID NO:9 with the exception that the MV Hpolypeptide has 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, or 19) of the following amino acid substitutions: S189, E235,N238, L249, G302, Y310, Q311, R377, M378, D416, N481, K488, G491, H495,D505, R533, S546, R547, and F552. In some cases, a modified Hpolypeptide provided herein can have the amino acid sequence set forthin SEQ ID NO:9 with the exception that the MV H polypeptide has 6 ormore (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)of the following amino acid substitutions: S189, E235, N238, L249, G302,Y310, Q311, R377, M378, D416, E471, N481, K488, G491, H495, D505, R533,S546, R547, and F552.

In some cases, a modified H polypeptide provided herein can include oneor more other amino acid modifications such as those described elsewhere(see, e.g., WO 2014/015242, Hu et al. (Virology, 192(1):351-4 (1993));Hummel and Bellini (J. Virol., 69(3):1913-16 (1995)); Rima et al. (J.Gen. Virol., 78:97-106 (1997)); Li and Qi (Arch. Virol., 147(4):775-86(2002)); Santibanez et al. (J. Gen. Virol., 86:365-74 (2005); and Taharaet al. (J. Virol., 82(9):4630-7 (2008)). For example, a modified Hpolypeptide provided herein also can include one or more of thefollowing amino acid substitutions: N238D, N282K, Y310C, N405S, D416N,K488E, S546G, R547G, and F552V. In some cases, a modified H polypeptideprovided herein also can include one or more of the following amino acidsubstitutions: N238D, N282K, Y310C, N405S, D416N, E471K, K488E, S546GR547G, and F552V. As another example, a modified H polypeptide providedherein can include SEQ ID NO:9 having an amino acid substitution at 6 ormore (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) ofthe following positions: S189, E235, N238, L249, G302, Y310, Q311, R377,M378, D416, N481, K488, G491, H495,

D505, R533, S546, R547, and F552. For example, a modified H polypeptideprovided herein can include SEQ ID NO:9 having 6 or more (e.g., 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following aminoacid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R,R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G, S546G,R547G, and F552V. As another example, a modified H polypeptide providedherein can include SEQ ID NO:9 having an amino acid substitution at 6 ormore (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)of the following positions: S189, E235, N238, L249, G302, Y310, Q311,R377, M378, D416, E471, N481, K488, G491, H495, D505, R533, S546, R547,and F552. For example, a modified H polypeptide provided herein caninclude SEQ ID NO:9 having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20) of the following amino acidsubstitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q,M378K, D416N, E471K, N481Y, K488E, G491E, H495R, D505T, R533G, S546G,R547G, and F552V.

Amino acid substitutions can be conservative or non-conservative.Conservative amino acid substitutions replace an amino acid with anamino acid of the same class, whereas non-conservative amino acidsubstitutions replace an amino acid with an amino acid of a differentclass. Examples of conservative substitutions include amino acidsubstitutions within the following groups: (1) glycine and alanine; (2)valine, isoleucine, and leucine; (3) aspartic acid and glutamic acid;(4) asparagine, glutamine, serine, and threonine; (5) lysine, histidine,and arginine; and (6) phenylalanine and tyrosine. In some cases, amodified H polypeptide provided herein can have the amino acid sequenceset forth in SEQ ID NO:3. In some cases, a modified MV H polypeptide canbe an H polypeptide from another member of the morbillivirus genus(e.g., CDV, CeMV, FeMV, PPRV, PDV, and RPV). For example, a modified MVH polypeptide can be a CDV H polypeptide (e.g., a polypeptide having thesequence set forth in GenBank Accession No. AAC26995 (VersionAAC26995.1; SEQ ID NO:8).

A nucleic acid encoding a modified F polypeptide provided herein canencode an F polypeptide that is heterologous to naturally occurring MV Fpolypeptides or to the F polypeptide having the amino acid sequence setforth in GenBank Accession No. AAF85672 (Version AAF85672.1, GI No.9181879; SEQ ID NO:4). Examples of naturally occurring MV F polypeptides(and the nucleic acid sequences encoding them) can be found in publicdatabase. For example, GenBank Accession No. KP205324 (VersionKP205324.1, GI No. 727347524; SEQ ID NO:5) provides an example of anucleic acid encoding a wild type F polypeptide. In some cases, a MV Fpolypeptide designed to be heterologous to naturally occurring MV Fpolypeptides and/or heterologous to the F polypeptide having the aminoacid sequence set forth in SEQ ID NO:4 can be referred to as a modifiedF polypeptide. The term “F polypeptide amino acid sequence” as usedherein refers to an amino acid sequence that is at least 85 percent(e.g., at least 85, 90, 95, 99, or 100 percent) identical to thesequence set forth in SEQ ID NO:4. In some cases, a modified MV Fpolypeptide can be an F polypeptide from another member of themorbillivirus genus (e.g., CDV, CeMV, FeMV, PPRV, PDV, and RPV). Forexample, a modified MV F polypeptide can be a CDV F polypeptide (e.g., apolypeptide having the sequence set forth in GenBank Accession No.ABR08390 (Version ABR08390.1, GI No. 148724186; SEQ ID NO:6) or GenBankAccession No. ABR08390 (Version ABO31365.1, GI No. 129770954; SEQ IDNO:7).

This document also provides recombinant viruses (e.g., MVs or Ads)containing a nucleic acid encoding a modified H polypeptide describedherein and a nucleic acid encoding a modified F polypeptide describedherein. A recombinant virus provided herein can be a chimeric virus. Insome cases, a recombinant virus can contain a nucleic acid encoding amodified MV H polypeptide described herein and a nucleic acid encoding amodified MV F polypeptide described herein. In some cases, a recombinantvirus can contain a modified MV H polypeptide described herein and amodified MV F polypeptide described herein.

In some cases, a recombinant virus provided herein can be amorbillivirus. Any appropriate morbillivirus can contain a nucleic aciddescribed herein (e.g., a nucleic acid encoding a modified MV Hpolypeptide and/or a nucleic acid encoding a modified MV F polypeptide).Species in the genus morbillivirus include, without limitation, MV (MV),canine distemper virus (CDV), cetacean morbillivirus (CeMV), felinemorbillivirus (FeMV), peste-des-petits-ruminants virus (PPRV), phocinedistemper virus (PDV), and rinderpest virus (RPV). In some cases, amorbillivirus provided herein is obtained from a MV. Examples of MVstrains include, without limitation, MVi/Madrid.SPA/50.10[H1],Edmonston, and Moraten vaccine.

In some cases, a recombinant virus provided herein can be an Ad. Anyappropriate Ad can contain a nucleic acid described herein (e.g., anucleic acid encoding a modified MV H polypeptide and/or a nucleic acidencoding a modified MV F polypeptide). In humans, species in the familyAdenoviridae include, without limitation, species A (AdA), species B(AdB), species C (AdC), species D (AdD), species E (AdE), species F(AdF), or species G (AdG).

A nucleic acid provided herein can be obtained using any appropriatemethod including, without limitation, common molecular cloning andchemical nucleic acid synthesis techniques. For example, PCR can be usedto construct nucleic acids that encode a modified H polypeptide or amodified F polypeptide provided herein. PCR refers to a procedure ortechnique in which target nucleic acid is amplified in a manner similarto that described in U.S. Pat. No. 4,683,195, and subsequentmodifications of the procedure described therein.

A nucleic acid provided herein can be incorporated into viruses bystandard techniques. For example, recombinant techniques can be used toinsert a nucleic acid encoding a modified H polypeptide or a modified Fpolypeptide provided herein into an infective viral cDNA. In some cases,a nucleic acid can be exogenous to a viral particle, e.g., an expressionvector contained within a cell such that the polypeptide encoded by thenucleic acid is expressed by the cell and then incorporated into a newviral particle (e.g., into the envelope of a new viral particle such asa recombinant virus).

Naturally occurring H polypeptides typically have receptor-binding andhemagglutination activities, and functionally cooperate with viral Fpolypeptides to induce fusion between target cells. Such fusion can bemediated through interactions between H polypeptides and receptors ontarget cells (e.g., CD46, SLAM, Nectin-4, desmoglein-2, or sialic acid).

Recombinant viruses (e.g., MVs or Ads) provided herein (e.g., containinga nucleic acid encoding a modified H polypeptide and a nucleic acidencoding a modified F polypeptide) can have reduced (or no)Nectin-4-dependent cell entry as compared to a virus having a naturallyoccurring H polypeptide and/or a naturally occurring F polypeptide. Forexample, when a modified H polypeptide and a modified F polypeptide areincorporated into a virus, the level of Nectin-4 dependent cell entryexhibited by the virus can be reduced (or eliminated) relative to thelevel of Nectin-4-dependent cell entry exhibited by a wild type viruscontaining a corresponding, naturally occurring H polypeptide. Amorbillivirus (e.g., a MV) containing a modified H polypeptide and amodified F polypeptide described herein can exhibit reduced (or no)Nectin-4-dependent entry into cells (e.g., epithelial cells) as comparedto the amount of Nectin-4-dependent entry of a non-modified MV-H intocells (e.g., epithelial cells). Cell entry via Nectin-4 can be assessedby standard techniques such as those described herein (see Example 1).Recombinant morbilliviruses provided herein can retain the ability tobind to CD46 and/or SLAM. Viruses containing a nucleic acid encoding amodified H polypeptide and a nucleic acid encoding a modified Fpolypeptide can therefore exhibit CD46- or SLAM-dependent cell entry,and cells containing such viruses can fuse in a CD46- or SLAM-dependentmanner. Cell entry via CD46 and/or SLAM receptors can be assessed bystandard techniques such as those described in WO 03/093431. In somecases, a recombinant morbilliviruses provided herein can retain theability to bind to CD46 and not SLAM. Viruses containing a nucleic acidencoding a modified H polypeptide and a nucleic acid encoding a modifiedF polypeptide can exhibit CD46-dependent cell entry, and cellscontaining such viruses can fuse in a CD46-dependent manner.

A virus provided herein can be attenuated. As used herein, the term“attenuated” refers to a virus that is immunologically related to a wildtype virus but which is not itself pathogenic. An attenuated MV, forexample, does not produce classical measles disease. Attenuated virusestypically are replication-competent, in that they are capable ofinfecting and replicating in a host cell without additional viralfunctions supplied by, for example, a helper virus or a plasmidexpression construct encoding such additional functions.

Any appropriate method can be used to identify a virus containing anucleic acid provided herein. Such methods include, without limitation,PCR and nucleic acid hybridization techniques such as Northern andSouthern analysis. In some cases, immunohistochemistry and biochemicaltechniques can be used to determine if a virus contains a particularnucleic acid by detecting the expression of a polypeptide encoded bythat particular nucleic acid.

Recombinant viruses (e.g., MVs or Ads) containing a nucleic acidencoding a modified H polypeptide and a nucleic acid encoding a modifiedF polypeptide provided herein can be used to treat cancer patients. Aparticular virus can be propagated in host cells in order to increasethe available number of copies of that virus, typically by at least2-fold (e.g., by 5- to 10-fold, by 50- to 100-fold, by 500- to1,000-fold, or even by as much as 5,000- to 10,000-fold). A virus can beexpanded until a desired concentration is obtained in standard cellculture media (e.g., DMEM or RPMI- 1640 supplemented with 5-10% fetalbovine serum at 37° C. in 5% CO₂). A viral titer typically can beassayed by inoculating cells (e.g., Vero cells) in culture. Virus can beharvested from infected cells by scraping cells from the dishes,subjecting them to freeze/thawing (e.g., approximately two rounds), andcentrifuging. The cleared supernatants represent “plaque purified”virus.

Viral stocks can be produced by infection of cell monolayers (e.g.,adsorption for about 1.5 hours at 37° C.), followed by scraping ofinfected cells into a suitable medium (e.g., Opti-MEM; Gibco/Invitrogen,Carlsbad, CA) and freeze/thaw lysis. Viral stocks can be aliquoted andfrozen, and can be stored at −70° C. to −80° C. at concentrations higherthan the therapeutically effective dose. A viral stock can be stored ina stabilizing solution. Stabilizing solutions are known in the art andinclude, without limitation, sugars (e.g., trehalose, dextrose,glucose), amino acids, glycerol, gelatin, monosodium glutamate, Ca²+,and Mg²+.

In some cases, recombinant viruses (e.g., MVs or Ads) containing anucleic acid encoding a modified H polypeptide and a nucleic acidencoding a modified F polypeptide provided herein can be used to treatcancer (e.g., to reduce tumor size, inhibit tumor growth, or reduce thenumber of viable tumor cells). As used herein, “reducing the number ofviable tumor cells” is meant to encompass (1) slowing the rate of growthof a population of tumor cells such that after a certain period of time,a tumor in a treated individual is smaller than it would have beenwithout treatment; (2) inhibiting the growth of a population of tumorcells completely, such that a tumor stops growing altogether aftertreatment; and/or (3) reducing the population of tumor cells such that atumor becomes smaller or even disappears after treatment.

Recombinant viruses (e.g., MVs or Ads) containing a nucleic acidencoding a modified H polypeptide and a nucleic acid encoding a modifiedF polypeptide provided herein can be administered to a cancer patientby, for example, direct injection into a group of cancer cells (e.g., atumor) or intravenous delivery to cancer cells. Types of cancer cellssusceptible to treatment with viruses include neuronal cells, glialcells, myelomonocytic cells, and the like. The methods provided hereincan be used to treat types of cancer that include, but are not limitedto, myeloma, melanoma, glioma, lymphoma, and cancers of the lung, brain,stomach, colon, rectum, kidney, prostate, ovary, and breast. Anattenuated MV containing a modified H polypeptide and a modified Fpolypeptide provided herein can be used to treat, for example, alymphoma (e.g., non- Hodgkin's Lymphoma).

A virus provided herein can be administered to a patient in abiologically compatible solution or a pharmaceutically acceptabledelivery vehicle, by administration either directly into a group ofcancer cells (e.g., intratumorally) or systemically (e.g.,intravenously). Suitable pharmaceutical formulations depend in part uponthe use and the route of entry, e.g., transdermal or by injection. Suchforms should not prevent the composition or formulation from reaching atarget cell (i.e., a cell to which the virus is desired to be deliveredto) or from exerting its effect. For example, pharmacologicalcompositions injected into the blood stream should be soluble.

While dosages administered will vary from patient to patient (e.g.,depending upon the size of a tumor), an effective dose can be determinedby setting as a lower limit the concentration of virus proven to be safeas a vaccine (e.g., 10³ pfu) and escalating to higher doses of up to10¹² pfu, while monitoring for a reduction in cancer cell growth alongwith the presence of any deleterious side effects. A therapeuticallyeffective dose typically provides at least a 10% reduction in the numberof cancer cells or in tumor size.

Escalating dose studies can be used to obtain a desired effect for agiven viral treatment (see, e.g., Nies and Spielberg, “Principles ofTherapeutics,” In Goodman & Gilman's The Pharmacological Basis ofTherapeutics, eds. Hardman, et al, McGraw-Hill, N.Y., 1996, pp 43-62).

Viruses provided herein can be delivered in a dose ranging from, forexample, about 10³ pfu to about 10¹² pfu (typically >10⁸ pfu). Atherapeutically effective dose can be provided in repeated doses. Repeatdosing is appropriate in cases in which observations of clinicalsymptoms or tumor size or monitoring assays indicate either that a groupof cancer cells or tumor has stopped shrinking or that the degree ofviral activity is declining while the tumor is still present. Repeatdoses (using the same or a different modified virus) can be administeredby the same route as initially used or by another route. Atherapeutically effective dose can be delivered in several discretedoses (e.g., days or weeks apart). In some cases, one to about twelvedoses can be provided. In some cases, a therapeutically effective doseof attenuated MV can be delivered by a sustained release formulation.

Viruses provided herein can be administered using a device for providingsustained release. A formulation for sustained release of a virus caninclude, for example, a polymeric excipient (e.g., a swellable ornon-swellable gel, or collagen). A therapeutically effective dose of avirus can be provided within a polymeric excipient, wherein theexcipient/virus composition is implanted at a site of cancer cells(e.g., in proximity to or within a tumor). The action of body fluidsgradually dissolves the excipient and continuously releases theeffective dose of virus over a period of time. In some cases, asustained release device can contain a series of alternating active andspacer layers. Each active layer of such a device typically contains adose of virus embedded in excipient, while each spacer layer containsonly excipient or low concentrations of virus (i.e., lower than theeffective dose). As each successive layer of the device dissolves,pulsed doses of virus are delivered. The size/formulation of the spacerlayers determines the time interval between doses and is optimizedaccording to the therapeutic regimen being used.

A virus provided herein can be directly administered. For example, avirus can be injected directly into a tumor (e.g., a lymphoma) that ispalpable through the skin. Ultrasound guidance also can be used in sucha method. In some cases, direct administration of a virus can beachieved via a catheter line or other medical access device, and can beused in conjunction with an imaging system to localize a group of cancercells. By this method, an implantable dosing device typically is placedin proximity to a group of cancer cells using a guidewire inserted intothe medical access device. An effective dose of a virus also can bedirectly administered to a group of cancer cells that is visible in anexposed surgical field.

In some cases, viruses provided herein can be delivered systemically.For example, systemic delivery can be achieved intravenously viainjection or via an intravenous delivery device designed foradministration of multiple doses of a medicament. Such devices include,but are not limited to, winged infusion needles, peripheral intravenouscatheters, midline catheters, peripherally inserted central catheters,and surgically placed catheters or ports.

The course of virus therapy can be monitored by evaluating changes inclinical symptoms (known in the art for each particular type of cancer)or by direct monitoring of the size of a group of cancer cells or tumor.A method for using a virus of the invention to treat cancer isconsidered effective if the cancer cell number, tumor size, tumorspecific antigen level, and/or other clinical symptoms are reduced by atleast 10 percent following administration of virus. For a solid tumor,for example, the effectiveness of virus treatment can be assessed bymeasuring the size or weight of the tumor before and after treatment.Tumor size can be measured either directly (e.g., using calipers), or byusing imaging techniques (e.g., X-ray, magnetic resonance imaging, orcomputerized tomography) or from the assessment of non- imaging opticaldata (e.g., spectral data). For a group of cancer cells (e.g., leukemiacells), the effectiveness of viral treatment can be determined bymeasuring the absolute number of leukemia cells in the circulation of apatient before and after treatment. The effectiveness of viral treatmentalso can be assessed by monitoring the levels of a cancer specificantigen. Cancer specific antigens include, for example, carcinoembryonicantigen (CEA), prostate specific antigen (PSA), prostatic acidphosphatase (PAP), CA 125, alpha-fetoprotein (AFP), carbohydrate antigen15-3, and carbohydrate antigen 19-4.

In some cases, recombinant viruses (e.g., MVs or Ads) containing anucleic acid encoding a modified H polypeptide and a nucleic acidencoding a modified F polypeptide provided herein can be used tovaccinate humans (e.g., infants less than 9 months of age or infantsless than 15 months of age). When vaccinating an infant less than 9months or 15 months of age using the MVs provided herein as a vaccine,the vaccine can effectively induce a protective immune response againstMV infection even though the infant contains maternal anti-MVantibodies. For example, a recombinant MV containing a nucleic acidencoding a modified H polypeptide and a nucleic acid encoding a modifiedF polypeptide can be used to stimulate an immune response against MV ina human.

Any appropriate patient can be treated using the materials and methodsdescribed herein. For example, a cancer patient treated or an infantvaccinated using a recombinant MV described herein can be a mammal(e.g., human, non-human primate, dog, and cat), bird, or reptile.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Recombinant MVs

A measles virus (MV#1) was produced to have a modified H protein (SEQ IDNO:3) having with the following amino acid substitutions (with respectto SEQ ID NO:1): H17S, D149N, S189P, G211S, E235G, N238D, S240N, L249P,L276G, V280I, N282K, G302R, E303G, Y310C, Q311R, Q334H, A359T, K364N,R377Q, M378K, P397L, N405S, D416N, T420A, V421A, L423P, F476L, N481Y,K488E, G491D, H495R, D505T, R533G, S546G, R547G, F552V, V562T, D574A,K576R, I594L, G603E, T609N, G613E, and T614A. This modified H protein(SEQ ID NO:3) was produced by introducing 19 point mutations into themeasles virus hemagglutinin protein (SEQ ID NO:9) of the wild-typeMVi/Madrid.SPA/50.10 (genotype H1) strain. The 19 point mutations wereS189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N,N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, and F552V.

A MV vaccine strain (MV #2) and the recombinant MV #1 were incubated for1 hour at 37° C. with mAb shown in FIG. 1 before infection of Vero/hSLAMcells seeded on a 96 well plate. The number of infected eGFP positivefoci were counted in quadruplicate 48 hours post-infection and expressedas percentage of the number of infected EGFP positive foci in absence ofnAb. MV #1 avoided neutralizing antibodies targeting multiple antigenicsites. H protein modifications that contributed to MV #1 escapingneutralization are shown in FIG. 1.

To determine the resistance of MV #1 to human anti-measles antiserum, MVglycoprotein-specific antibodies (e.g., H-reactive and F-reactive)antibodies were depleted from measles-immune human serum. Serum sampleswere diluted 1:10 in culture medium (RPMI 1640 (CORNING, Manassas, VA,USA) without fetal bovine serum (FBS)) and subsequently cultured for 4days onto a monolayer of Mel-JuSo cells either expressing or notexpressing MV glycoproteins. Supernatants were collected and tested in a1:100 final dilution for the presence of H- or F- or VCA-specificantibodies by a FACS-measured immunofluorescence assay, using the stablytransfected human melanoma cells Mel-JuSo/MV-H or Mel-JuSO/MV-F astarget cells. Epstein-Barr virus (EBV) VCA-specific antibodies werequantified by a commercial ELISA (IBL International, Hamburg, Germany).MV #1 was efficiently neutralized by F-reactive antibodies inmeasles-immune human serum. A fluorescence plaque reduction microneutralization assay was used to determine anti-MV-H immunity inducedupon vaccination. MV #1 was resistant to the H reactive component of thehuman anti-measles antibody response.

To confirm that apart from MV H protein-specific antibodies, MV Fprotein-specific antibodies are also important for MV neutralization,another wild-type Edmonton strain measles virus (MV #3) was produced byreplacing the MV F protein of a wild-type Edmonston strain MV with awild-type Ondersterpoort strain CDV F protein. Two other measles viruses(MV #4 and MV #5) were produced. MV #4 was produced by replacing the MVH protein of a wild-type Edmonston strain MV with a wild-type CDV Hprotein. MV #5 was produced by replacing the MV F protein of MV #4 witha wild-type (i.e., Ondersterpoort strain) CDV F protein. Schematicrepresentations of MV #3, MV #4, and MV #5 viruses are shown in FIG. 2.The MV #2, MV #3, MV #4 and MV #5 viruses were tested as follows.Vero/cSLAM cells were infected with the different viruses andmicrophotographs were taken 48 hours post-infection. A neutralizationassay was performed and revealed that both MV H and MV F elicitneutralizing antibodies.

A productive heterotypic fusion of CDV Ondersterpoort and MVH #1. CHOcells expressing or not the MV receptors were seeded in a 24 well plate,and were cotransfected with plasmids encoding MVH #1 (1 μg) and Fproteins (1 μg; MV vaccine strain, MVF; Ondersterpoort vaccine strain,CDV F). Syncytium formation was evaluated 24 hours later. Syncytiumforming activity results demonstrated that CDV F can trigger fusion withno impaired in syncytium formation when coexpressed with MVH #1 directedmanner. A virus encoding both MVH #1 and CDV F was rescued and confirmthe monotropism for cells expressing CD46.

Surface expression patterns for the MV receptors were examined usingflow cytometry. Two days after infection, productive infection wasevaluated under a microscope. These results demonstrated that MVencoding MV #1 and CDV F propagated efficiently on cells expressing theCD46 receptor.

C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, EEUU) were immunizedvia hydrodynamic delivery with pCG plasmid (5 μg) encoding for MVHprotein from MV #1. The neutralization capacity of the antibodiesgenerated evaluated 4 weeks later after blood collection from thejugular vein. Mice sera were heat inactivated and serially dilutedeither in Opti-MEM. Equal volume of respective virus encoding differentMVH gene proteins at 30 PFU/well were mixed with the respectivepolyclonal antibodies at 2-fold serial various dilutions in 96-wellplates (Costar Corp., Cambridge, MA, USA), incubated at 37° C. for 1hour and inoculated onto 80 to 90% confluent Vero/hSLAM cells. eGFPautofluorescence was visualized under a fluorescent microscope after 2days of culture and a neutralization titer was given as the higherdilution blocking 100% of viral infectivity. These results demonstratedthat MV #1 provoked an H glycoprotein-directed neutralizing antibodyresponse that cross-neutralizes wild type strains of MV.

Example 2—Additional Recombinant MVs

For this Example 2, MV is referred to as MeV; MV #1 is referred to asMeVΔ7, Δ7, or Δ8; MV #2 is referred to as MeV #1; MV #3 is referred toas Virus 3; MV #4 is referred to as MeV #4; and MV #5 is referred to asMeV #2. The synthesis of several of these were described again inExample 2, and some of the data presented in Example 1 also arepresented in Example 2. In addition, MeVΔ7 was used to generate the Δ8virus.

Cells and Viruses

Vero cells (CCL-81, ATCC), the stably transfected Vero human(Vero/hSLAM) (Ono et al., J. Virol., 75(9):4399-401 (2001)) and dog(Vero/dogSLAM) (von Messling et al., J. Virol., 77(23):12579-91 (2003))SLAM were grown in in Dulbecco's modified minimal essential medium(DMEM) (HyClone, GE Healthcare Life Science) supplemented with 5%(vol./vol.) heat-inactivated fetal bovine serum (FBS) (Gibco) and 0.5mg/mL of Geneticin (G418; Corning) (Vero/hSLAM) or 1 mg/mL Zeocin(ThermoFisher, Walthman MA) (Vero/dSLAM). Chinese hamster ovary (CHO)cells, CHO-CD46 (Nakamura et al., Nat. Biotechnol., 22(3):331-6 (2004)),CHO-SLAM (Tatsuo et al., Nature, 406:893-6 (2000)), and CHO-N4 (Liu etal., J. Virol., 88(4):2195-204 (2014)) were cultured as described. BabyHamster Kidney cells (BHK) were maintained in DMEM-10% FBS. Viruses werepropagated as described elsewhere (Munoz-Alia et al., J. Virol.,91(11):e00209-17 (2017); Munoz-Alia et al., PLoS One, 13(2):e0192245(2018)).

Constructs and Rescue of Recombinant MeVs

The recombinant MeV was based on the molecular cDNA clone of theMoraten/Schwartz vaccine strain contained in p(+)MeV^(vac2)(EGFP)Nplasmid (del Valle et al., J. Virol., 81(19):10597-605 (2007)). In thisplasmid, the enhanced green fluorescent protein (EGFP) was insertedupstream the N gene. To avoid plasmid instability upon its propagationin bacteria, the plasmid backbone was replaced by the pSMART®LCkanvector (Lucigen, Middleton, WI) in a stepwise manner following twoapproaches. In the first approach, a multi-cloning site comprising SacIland NotI restriction enzymes were added to the vector. Then, an optimalT7 promoter followed by a hammerhead ribozyme (HHrbz) (FIG. 7) wasinserted upstream the viral genome by insertion of the sequence directlyinto the forward primer, amplifying the MeV genome up to a uniqueinternal restriction site SacIl located at the beginning of the P gene.The SacII-NotI fragment in p(+)MV^(vac2)(EGFP)N plasmid was theninserted into the equally digested pSMART®LCkan vector. In the secondapproach, a cassette containing the human Elongation Factor-1α corepromoter, chimetric intron, T7 RNA polymerase promoter, HHrbz andcloning sites was synthesized and ligated into the vector. All plasmidpropagations were performed in Escherichia coli Stb12™ cells(Invitrogen, 10268019) grown in at 30° C.

To produce envelope-exchange MeVs, the CDV Ondersterpoort vaccine strainH (CDV-H) and F (CDV-F) genes originally contained in pCG plasmid (vonMessling et al., J. Virol., 75(14):6418-27 (2001)) were used. To replaceMeV-H from the MeV backbone, site-directed mutagenesis (QuikChangesite-directed mutagenesis kit, Agilent) was first used to removed a SpeIsite in CDV-H, and a Y537D substitution was then introduced to reducebinding by cross-reactive neutralizing antibodies in human sera (Zhanget al., Virology, 482:218-24 (2015)). Pad and SpeI restriction sites(underlined) were introduced into the beginning and end of the gene,respectively, by polymerase chain reaction using forward primer5′-ttaattaaaacttagggtgcaagatcatcgataatgctcccctaccaagacaagg-3′ andreverse primer5′-actagtgggtatgcctgatgtctgggtgacatcatgtgattggttcactagcagccttaatggtggtgatggtggtggctcccccttgcggccgcggccggctgggccgctctaccctcgatacggttacatgagaatcttatacggac-3′,leaving the untranslated region (UTR) unchanged. The PCR product wasdigested with PacI and SpeI and cloned into the MeV backbone. To replaceMeV-F from MeV antigenome plasmid, pCG-CDV-F was digested with HpaI/SpeIand inserted into equally digested pCG-MeV-F. The NarI/SpeI fragment ofthis plasmid was then used to replace that of MeV.

The recovery of recombinant MeVs (rMeV) was performed by co-transfectionof rMeV antigenomic plasmid construct, N, P, and L supporting plasmidsderived from a MeV isolate genotype B3.1 (Munoz-Alia et al., Virus Res.,196:122-7 (2015)), and a codon-optimized T7 RNA polymerase (obtainedfrom Behur Lee, Addgene plasmid 65974), with Lipofectamine LTX/PLUStransfection reagent (Invitrogen). Transfected cells were co-culturewith Vero/hSLAM cells and the virus was amplified thereafter. Theidentity of the recombinant MeVs was confirmed by Sanger sequencingafter RNA extraction from infected cells.

Fusion Assay

Cells (5×10⁵/well in 6-well plate) were co-transfected using Fugene HD(Promega) with (1 μg) of pCG plasmid encoding for vaccine strain MeV-Fand pCG encoding the appropriate MeV-H. Fusion activity was evaluated 24hours later after Hema-Quik staining (Fisher Scientific 123-745).

To quantify cell fusion, the dual-split luciferase assay was used asdescribed elsewhere (Saw et al., Methods, 90:68-75 (2015)). Briefly,effector BHK cells (3×10⁴) in a black 96-well plates were co-transfectedwith 33 ng each of the MeV-H and MeV-F expression plasmids and one ofthe split luciferase plasmids, DSPs-11 (obtained from Z. Matsuda). As acontrol, only the MeV-F and DSPs-11 plasmids were transfected. 2×10⁵cells per well in 6-well plates of target cells, CHO cells, and CHOcells expressing the respective measles virus receptors were transfectedwith 1.5 μg of the other dual-split-reporter plasmid (DSPs₁₋₇). 24 hoursafter transfection, target cells were detached with Versene (LifeTechnologies) and co-cultured with the effector cells in Fusion media(DMEM-F12 without Phenol Red+40 mM HEPES), supplemented with 1:1000dilution of the cell permeable luciferase substrate EnduREN (Promega).Luminescence resulting from cell fusion and mixing of cytoplasmiccontent between target and effector cells was monitored with a TopcountNXT Luminometer (Packard Instrument Company, Meriden CT) at theindicated time points. The data represented the mean and standarddeviation of three replicates for each H plasmid.

FACS Analysis and Quantification of Cell Surface Molecules

Cells were washed and detached by using Versene (Gibco) and immediatelyincubated with phycoerythrin-conjugated antibodies anti-SLAM (FAB1642P;R&D Systems), anti-CD46 (FAB2005P; R&D Systems), and anti-nectin-4(FAB2659P; R&D Systems), or with control isotype antibody (IC0041P; R&DSystems). After incubation for 1 hour at 4° C., cells were washed again,and fluorescence was measured in a FACSCanto flow cytometry system (BDBioscience). The number of receptors per cell was estimated in referenceto calibration beads (BD QuantiBrite; BD Biosciences).

Recombinant Proteins and Binding Assays

Coding sequence of CD46 ectodomain (residues 35-328) was amplified viaPCR from pGEM-CD46 vector (Sino Biologicals Inc., HG12239-G) andinserted into pFUSE vector (pfcl-hgle3; Invivogen) in frame with themurine Ig κ-chain leader sequence and a 3C protease cleavage sequence atthe 5′-end of the Fc region using In-Fusion cloning kit (Clontech).CD46-Fc, SLAM-Fc, and nectin-4-Fc (Munoz-Alia et al., PLoS One,13(2):e0192245 (2018)) recombinant proteins were expressed in Expi293cells (Gibco) and purified from culture supernatant as describedelsewhere (Munoz-Alia et al., PLoS One, 13(2):e0192245 (2018)). Theexpression and purification of recombinant soluble MeV-H were performedas described elsewhere (Munoz-Alia et al., J. Virol., 91(11):e00209-17(2017)). Binding of the receptors-Fc to MeV-H was determined byenzyme-linked immunosorbent assay as described elsewhere (Munoz-Alia etal., PLoS One, 13(2):e0192245 (2018)). The absorbance at 450 nm wasmeasured on an Infinite M200Pro microplate reader (Tecan). The data wereanalyzed using Prism software (GraphPad) and adjusted to a one-sitebinding saturation mode to determine the half-saturating concentration(apparent Kd values [dissociation constant]). Values reported exhibitedan excellent fit (R²>0.99).

Virus Protein Content

Virus preps were heated in the presence of DTT, fractionated into 4-12%Bis-Tris polyacrylamide gel, and transferred to PDVF membranes. Blotswere then analyzed with anti-MeV-Hcyt, anti-MeV-N, anti-MeV-F, andanti-GFP probed with conjugated secondary rabbit antibody (ThermoFisher,#31642). The blots were revealed with SuperSignal Wester PicoChemiluminescent Substrate (ThermoFisher) and analyzed under a ChemiDocImaging Sytem (BIO-RAD).

Serologic Assays

Virus neutralization assay was performed based on the fluorescence-basedplaque reduction microneutralization (PRMN) assay as described elsewhere(Munoz-Alia et al., J. Virol., 91(11):e00209-17 (2017); Munoz-Alia etal., PLoS One, 13(2):e0192245 (2018); and Munoz-Alia et al., Virus Res.,236:30-43 (2017)). Each assay was repeated at least two times indifferent days with four replicates per assay. Fifty percent inhibitoryconcentration (IC₅₀) was calculated after fitting the data to asigmoidal dose-response (variable slope) with GraphPad software (Prism7).

Rabbit anti-MeV-H antisera were generated by immunization withadenovirus expressing the MeV-H from the vaccine strain (Lech et al.,PLoS One, 8(1):e52306 (2013)).

The following reagents were obtained through BEI Resources, NIAID, NIH:Polyclonal Anti-Canine Distemper Virus, Lederle Avirulent (antiserum,Ferret), NR-4025; and polyclonal Anti-Measles Virus, Edmonston,(antiserum, Guinea pig), NR-4024.

Murine monoclonal anti-hemagglutinin antibodies were produced andcharacterized as described elsewhere (Muñoz-Alia et al., Virus Research,e00209-17 (2017); Ziegler et al., J. Gen. Virol., 77(Pt 10):2479-89(1996); Fournier et al., J. Gen. Virol., 78:1295-302 (1997); Ertl OT.Immunodominant regions and novel functional domains on the measles virushemagglutinin protein. Germany: Eberhard Karls University; 2003; Hu etal., Virology, 192:351-4 (1993); and Masse et al., J. Virol.,78(17):9051-63 (2004)). Polyclonal antibodies were generated by viagene-based hydrodynamic injection (Liu et al., Gene Ther., 6(7):1258-66(1999)) of C57BL/6 mice with 20 μg of plasmid DNA.

Human serum was collected from the Erasmus MC' serum bank from healthysubjects aged 17-18 (de Swart et al., J. Virol., 79(17):11547-51(2005)). They were likely never exposed to wild-type MeV and received amonovalent measles vaccination at the age of 14 months and ameasles-mumps-rubella vaccination at the age of 9 years. All polyclonalsera and ascites containing mAb were heat inactivated (30 minutes, 56°C.) before testing.

The Epstein-Barr virus (BCA) IgG titer was determined by a commerciallyavailable assay (IBL International GMbH, cat. No. 57351). The assay fordetermining MeV-specific IgG levels was performed as described elsewhere(de Swart et al., J. Virol., 79(17):11547-51 (2005); and de Swart etal., J. Virol. Methods., 71:35-44 (1998)).

Structural Modeling

A model of the MeV-H Stealth was generated at >90% confidence using theprogram Phyre2 (Kelley et al., Nat. Protoc., 10(6):845-58 (2015)). Thestructure was then submitted for in silico glycosylation using theGlyPro server (http://www.glycosciences.de), which produced a complexpenta-antennary N-glycan model at all predicted N-glycosilation sites,including N168 and N187 that are part of disordered regions. The CD46receptors from the MeV-H/CD46 crystallographic co-structure (PDB 3INB)was superimposed and manipulated using PyMOL software(http://pymol.org).

Statistical Analysis

Statistical significance was calculated with GraphPad Prism 7 followingthe appropriate statistical test.

Results Modeling an Antigenic Drift into MeV-H

The MeV-H has seven major antigenic sites, and multiple disruptions ofup to four of those sites does not abrogate polyclonal antibodiesneutralization (Munoz-Alia et al., J. Virol., 91(11):e00209-17 (2017);Lech et al., PLoS One, 8(1):e52306 (2013); and Munoz-Alia et al., PLoSOne, 13(2):e0192245 (2018)). It is possible that due to a lack of B cellimmunodominance, ablation of all antigenic sites can generate anon-neutralizable variant. To explore this, all epitopes described forMeV-H were systematically disrupted. The experimental design was basedon incorporating spontaneous neutralizing mAb escape-mutant selectionsonto the MeV-H background of genotype H1. This particular strain waschosen based on a previous observation of being one of the mostantigenically advanced MeV-H (Munoz-Alia et al., PLoS One,13(2):e0192245 (2018)), which would enable one to minimize thealteration of the otherwise rigid MeV-H protein (Fulton et al., CellRep., 11(9):1331-8 (2015)). A list of nAb binding regions was generated,and diruptions of those regions were combined into a single MeV-H, nameherein 47 (also known as MV #1 in Example 1)(Table 3).

TABLE 3 Antigenic Alternative mAb Refer- site names Substitutionresistance ences Φ E1 N282K BH015, BH130 B Ia I, VI, E4, E235G E185 CLE, V G302R E39 C Y310C BH038, BH141, A I-29 Q311R E103 C Ib NE, IVL249P BH047, BH059, D BH129 IIa II, SSE, VII E488K BH097 E G491D 16CD11F IIb II, SSE, VII 416DLS→NLS E128 C BH125 B III III, IIIA, S189P I-44 FIIIB, VII, D505T 80-II-B2 G RBE, E2 R533G Cl55 H 16DE6 F R547G 20H6 EF552V I-41 F R377Q, L77 I M378K IV ‘noose’, P397L BH006, BH216 (54) HNE,I, E3, N405S 8905 H D, D/E E471K BH030 Example 2 herein For references:A = Lech et al., PLoS One, 8(1): e52306 (2013). B = Munoz-Alia et al.,PLoS One, 13(2): e0192245 (2018). C = Tahara et al., J. Virol., 87(1):666-75 (2013). D = Munoz-Alia et al., J. Virol., 91(11): e00209-17(2017). E = Lech et al., Virology, 454-455: 237-46 (2014). F = Hu etal., Virology, 192(1): 351-4 (1993). G = Hummel et al., J. Virol.,69(3): 1913-6 (1995). H = Muñoz-Alía et al., Virus Research, 236: 30-43(2017). I = Liebert et al., J. Virol., 68(3): 1486-93 (1994). J =Finsterbusch et al., J. Gen. Virol., 90: 2739-45 (2009).

Tropism Engineering into MeV-H

Since mutations in antigenic site III (receptor binding site, RBS) wereincompatible with wild-type tropism (SLAM and nectin-4), the plan was toswitch receptor-specificity towards CD46 via a number of amino acidsubstitutions: N481Y (Lecouturier et al., J. Virol., 70(7):4200-4(1996)), H495R (Okada et al., J. Virol., 83(17):8713-21 (2009)), andS546G (Shibahara et al., J. Gen. Virol., 75:3511-6 (1994)). To evaluatethe impact of the amino acid substitutions in the receptor-dependentfusion activity, transient expression of the MeV-H mutants was performedin combination with the vaccine-derived MeV-F. The correspondingrecombinant MeV were also rescued by reverse genetics since cell-celltransmission can occurred in the absence of obvious syncytium formation(Langedijk et al., J. Virol., 85(21):11242-54 (2011)). The results weredisplayed in FIG. 8. When MeV-H vaccine strain (A) was used, both virusentry and cell fusion were observed in CHO cells expressing either SLAM,CD46, or nectin-4. Similarly, MeV-H H1 allowed for virus entry andsyncytia formation in SLAM and nectin-4-expressing cells. Theintroduction of N481Y, H495R, or H495R/S546G in the latter backgrounddid not significantly increase CD46-dependent fusion activity as seen bytransient transfection assays. However, virus entry was observed in theabsence of syncytium formation for the N481Y and H495R/S546G mutants.The inclusion of H495R in the context of N481Y mutant restored thelevels of CD46-dependent fusion to those observed by MeV-H A. Theaddition of S546G in this mutant enhanced almost 2-fold theCD46-dependent fusion, and a similar increase was observed with theN481Y/S546G mutant. Nevertheless, CD46-dependent enhancement infectionwas observed only when the triple mutant (N481Y/H495R/S546G) was used,and this combination was therefore chosen as background of nAb-escapemutations. MeV-H can systematically resist neutralization by 30 knownmurine monoclonal antibodies

Table 3 was initially used as a foundation to generate a MeV-H globulardomain escape virus. Using this information, together with the tripleCD46-tropism substitutions, a Δ7 virus was directly engineered, in whichall seven operationally non-ovelapping antigenic sites so far describedare disrupted (Φ, Ia, Ib, IIa, IIb, III, and IV) (FIG. 9A). To determinewhether the number of mutations introduced was suffice to abrogatedneutralization of other nAbs specific for each site, the neutralizationsensitivity of viruses possessing MeV-H A, H1 and Δ7 was determinedagainst a panel of 30 mAbs. The results were summarized in FIG. 9B,showing that A viruses were neutralized by all 30 nAbs tested whereasthis number was reduced to 18 for H1 viruses. On the other extreme, theΔ7 viruses were only neutralized by nAb BH030.

The fact that both Δ7 and H1 viruses were neutralized similarly by BH030indicated that the mutations introduced in Δ7 did not eliminated thisnAb epitope. However, both viruses showed a 18-fold reduction insensitivity to neutralization when compare to A viruses (IC₅₀ of 1312ng/mL versus 71.6 ng/mL).

The following was performed to determine whether this phenotype wasapplicable to other wild-type-specific MeV-H proteins or whether it wasa peculiarity of the H1 genotype background used. Neutralizationanalysis indicated that H1 viruses possessed indeed some resistanceagainst neutralization by BH030 in comparison with A, B3.1, C1, D4, D6,D7, D8, D9, F, and G viruses. However, C2 viruses did show a completelack of neutralization sensitivity (FIG. 10A). Based on the sequenceanalysis of the amino acid sequence and molecular structure of MeV-H, aE471K mutation was identified as a candidate for nAb resistance (FIG.10B). To confirm these predictions, the E417K mutation was inserted intothe Δ7 virus (this new virus was termed the “Δ8 virus”), and the 48virus's neutralization sensitivity to nAb BH030 was assessed. Differentfrom 47 viruses, Δ8 viruses displayed full escape from BH030-mediatedneutralization (FIG. 10C). Interestingly, E471 lies within the regionassigned to antigenic site IIb (II in Tahara et al., J. Virol.,87(1):666-75 (2013) or “sugar-shielded epitope” in Tahara et al.,Viruses, 8(8): pii:E216 (2016)), which is defined for being masked by anN416-linked sugar present in some genotypes (FIG. 10D). To confirm thata N416-attached sugar does not protect from BH030 neutralization, theneutralization sensitivity of a D416N mutant A virus (Munoz-Alia et al.,PLoS One, 13(2):e0192245 (2018)) was tested. Given that Δ7 viruses alsopossessed a N416 sugar, no resistance to neutralization was observedsuggesting that BH030 might target a new operationally non-overlappingantigenic site (designed herein as V, which presumably expands betweenantigenic sites IIb and III (FIG. 10D). These results showed thatmutations of key residues in major antigenic sites can be combined toresult in neutralization escape of large panel of 30 neutralizingantibodies.

Epitope Elimination into MeV-H Abolishes Cross-Neutralization

The following was performed to determine whether the B-cell epitopedisruption in MeV-H affected its antigenicity. Mice were given ahydrodynamic injection of MeV-H encoding plasmid, and the antibodyresponses were evaluated one month later (FIG. 11A). Neutralizationassays showed that mice immunized with MeV-HΔ7 had lower titer ofneutralizing antibodies than MeV-HA but did not reach statisticalsignificance, likely due to variability (FIG. 11B). MeV-I-148 wasnonetheless unable to generate any detectable levels (FIG. 11C). Thefollowing was performed to assess whether MeV-HΔ8 was generatingnon-cross reactive antibodies against new epitopes by probing theneutralization activity of Δ8 viruses themselves. The results shown inFIG. 11C showed that Δ8 viruses were neutralized by homotypicantibodies, i.e., antibodies triggered by immunization with MeV-H Δ8, asit was by anti-MeV- H H1 and Δ7, for having epitopes in common butdistinct from MeV-H A. Those neutralizing antibodies induced by MeV-HAimmunization exerted no neutralizing activity against Δ8 viruses. Then,elimination of multiple antigenic sites abolishes cross-neutralizationand allows the virus to escape an anti-MeV-H polyclonal response.

Δ7 Viruses can Escape Polyclonal Measles Vaccine-Induced NeutralizingAntibodies if Devoided of MeV-F-Specific Antibodies

Different animal models might exhibit a different antibody repertoire(Nachbagauer et al., Nat. Immunol., 18(4):464-73 (2017)). The breath ofantibodies arisen in rabbits after MeV-H A immunization was assessed vianeutralization analysis. Although virus Δ8 displayed a trend towardsneutralization (FIG. 12A), it exhibited a 8-fold (3 log₂) reduction inND₅₀ titer in comparison with the vaccine virus. Since a 4-folddifference (2 log₂ or antigenic units (Smith et al., Science,305(5682):371-6 (2004)) or higher warrants an update of the humanseasonal influenza vaccine (Russell et al., Vaccine, 26(Suppl 4):D31-4(2008); and Garten et al., Science, 325(5937):197-201 (2009)), virus Δ8was considered significantly antigenically different from the vaccinevirus. On the contrary, the immediate precursor virus (Δ7) wasantigenically indistinguishable as the parental precursor was (H1).Because a K471E mutation distinguishes Δ7 from Δ8 viruses and led toantigenic variation, there was a desire to assure that the antigenicdifferences were the results of the combination of all mAb-escape mutantselections and not to the existence of dominant mutants. Thus, aprevious panel of recombinant MeV possessing other genotype-specificMeV-H gene proteins were tested (Munoz-Alia et al., J. Virol.,91(11):e00209-17 (2017); and Munoz-Alia et al., PLoS One, 13(2):e0192245(2018)). Of particular interest was the MeV C2 since it possessed theK471E mutation. However, the differences in the PRMN titers were lessthan 2-fold across the board and therefore considered insignificant.Overall, these results suggest that antigenic variation in measles virushas both an incremental and a pulsed component; i.e. beyond an antigenicthreshold, amino acid substitutions might have an accumulative effect.

Since Δ7, but not Δ8, viruses could potentially be used as a pre-vaccinein infants with maternal antibodies, tests were performed to testwhether the antigenic variation in Δ7 could lead to a reduce recognitionby sera from patients having received the measles vaccine. Thisinitially involved selecting for testing six sera samples from Dutchindividuals aged 17 to 23 at the time of serum collection. Based on theneutralization titer and the record of measles outbreak in theNetherland, the human samples likely correspond to two-dose recipientsof the measles vaccine. Δ7 virus and vaccine strain virus were tested byPRMN with human sera #126, #128, #129, #134, #136, and #137. The meanND₅₀ titer for the Δ8 virus was 1.41-fold lower (0.50 antigenic units)than that of the homologous titer of the vaccine strain, indicating alack of antigenic variation between the two viruses. A correlation wasfound between MeV-H and MeV-F-specific antibodies (Pearson R=0.54,p<0.05). To test whether MeV-F specific were masking a potentialantigenic variation, MeV-F specific antibody were depleted, and theassay was repeated. As illustrated in FIG. 12B, incubation of the humansera with mock transfected cells (condition 1) resulted in no decreaseof the MeV-F specific IgG antibodies in comparison with the untreatedsample (condition 0). Conversely, incubation with MeV-F-expressing cellsresulted in the depletion of MeV-F-specific antibodies, whilstMeV-H-specific antibody levels remained unchanged. To account for smalldilution factors introduced upon difference depletion conditions,Epstein Barr VCA IgG antibodies, broadly prevalent in the humanpopulation, were additionally tested, and they were used as control ofthe level of antibodies used thereof (FIG. 12C). Then, both undepletedand MeV-F-depleted human sera were re-tested for their neutralizationcapacity. Whereas MeV-F-depleted human sera lost insignificantlyneutralization potency against the vaccine virus (1 antigenic unit), thereduction was now significant (7-fold) when 47 viruses were comparedwith the vaccine virus (FIG. 12D).

These results indicate that vaccination in humans induce a narrowerneutralizing antibody response than rabbits, and Δ7 viruses couldpotentially close the gap of vaccination for young infants if devoid ofanti-MeV-F antibodies.

MeV-H and MeV-F Elicit Neutralizing Antibodies

Neutralizing antibodies against the MeV-F in measles-immune human seracan buffer the effect of accumulative antigenic substitutions in MeV-H.To further gain insights into the contribution of the two MeVglycoproteins to virus neutralization, a two-fold approach was used: 1)Depletion MeV-H and MeV-F-specific antibodies (FIG. 13A) and 2) Study ofthe neutralization sensitivity of an isogenic set of viral chimera withthree different glycoprotein-exchanges. This envelope-exchange virus hadall genes derived from MeV with the exception of H and F, which wereeither singly and doubly exchanged with the related but non-crossreactive Canine Distemper Virus (CDV) H and F (Miest et al., Mol. Ther.,19(10):1813-20 (2011); and Zhang et al., Virology, 482:218-24 (2015)).Thus, double switching of MeV-H and MeV-F protein genes for those fromCDV generated MeV #2, whereas a single switch in either MeV-H or MeV-Fproduced MeV #3 and MeV #4, respectively (FIG. 13B). The CDV-H proteingene used possessed an intentional Y537D substitution apropos theOndersterpoort vaccine strain, since it was shown to reduce potentialcross-neutralization of CDV by human sera (Munoz-Alia et al., J. Virol.,91(11):e00209-17 (2017); Zhang et al., Virology, 482:218-24 (2015)). Theparental MeV (MeV #1) as well as all three chimeras (MeV #2, MeV #3, MeV#4) formed indistinguishable syncytia in Vero cells, demonstratingheterotypic complementation (FIG. 13B).

For the MeV H or MeV-F-specific antibodies, a commercially availablepool of human sera composed from approximately 60 to 80 American donorswas used. Due to the high antibody titers in this pool, it is likely tohave been induced mainly by exposure to wild-type viruses (Itoh et al.,J. Clin. Microbiol., 40(5):1733-8 (2002)). FIG. 13A illustrates thedepletion process. Serum absorption with MeV-H-expressing cells(condition 2) completely removed all human serum binding activity toMeV-H protein, whilst MeV-F specific antibody levels remainedunaffected. In contrast, absorption with MeV-F (condition 3)specifically removed human serum binding to MeV-F whilst MeV-H-specificantibodies remained unaffected. Serum absorption with the parental cellline resulted in no decreased of the human serum binding to both MeV-Hand MeV-F in comparison to the original human serum material (condition1 and 0, respectively).

The neutralization potency of MeV-H and MeV-F reactive antibodies wasthen measured in measles-immune human. The PRMN assay showed thatabsorption of either MeV-F or MeV-H component did not substantiallyaffect neutralizing activity from human sera against MeV (MeV #1). Onthe other hand, serum absorption with both MeV-F and MeV-H resulted inthe complete loss of neutralizing activity from human sera. Thus, bothMeV-H and MeV-F-specific antibodies were equally important forneutralizing MeV. As expected, the measles-immune human sera did notshow neutralizing activity against MeV #2, regardless of the seratreatment. On the other hand, MeV #1, MeV #3 and MeV #4 were efficientlyneutralized without distinction by non-absorbed sera. Concerning theviral chimera with simple exchanges (MeV #3 and MeV #4), only when theMeV-specific antibody component that matched that present in the viruswas depleted, did they show resistance to neutralization. These resultsindicate that both MeV-F and MeV-H are immunogenic and they cooperate tobuffer antigenic variations.

Antigenic Novelty does not Sacrifice Fitness

We next hypothesized that the breadth of antibody response against bothMeV envelope glycoproteins harbors the antigenically staticity of MeV.In an attempt to address this hypothesis, the rescue of Δ8 viruses waspursued in combination with the heterotypic CDV-F, as a proxy for afully antigenically distinct virus (this virus was referred to asStealth going forward). MeV Stealth was not obtained until therobustness of the MeV rescue system was improved. Different from therescue of the parental recombinant MeV Moraten vaccine, MeV Stealth wasisolated and expanded from a single GFP-positive cell observed aftermultiple independent rescue attempts. After five semi-blind passages,the virus was able to spread through the cell monolayer (FIG. 7). Thevirus was further propagated to produce virus stock and tested forSanger sequencing and western-blot analysis. Immunoblotting of purifiedvirions demonstrated that Stealth lacked the homotypic MeV-F protein andshowed otherwise a similar protein content to the vaccine strain (FIG.14B). Moreover, sequencing results showed no compensatory mutation ineither of the glycoproteins-coding sequence, further confirming theviability of a Stealth virus encoding MeV-HΔ8 in combination with CDV-F.

To determine whether MeV Stealth associated a fitness trade-off, thegrowth kinetic of MeV Stealth was examined in cultured cells. Thosekinetics were compared with those of recombinant MeVs possessing MeV-HA, MeV-H H1, and MeV-M.8. MeV A replicated to higher titers at 12 and 48hpi than Stealth and Δ8 viruses (FIG. 14A). These two viruses showed a24 hour lag titer peak. MeV H1 replicated to lower titers than any ofthe other viruses thorough the time course. Since MeV Δ8 possessed aMeV-H derived from MeV-H H1, these results indicated that the mutationsintroduced into MeV-H cooperate for a better complementation withheterologous F proteins. Next, 15 human sera samples were used todetermine whether Stealth virus was indeed resistant to neutralizationby human antibodies triggered by the measles vaccine (virus A). Thepooled human sera used before was avoided since high titers areindicative of different exposure histories to measles virus, which mighthave induced different repertoires of genotype-specific neutralizingantibodies (de Swart et al., J. Gen. Virol., 90:2982-9 (2009); Tamin etal., J. Infec. Dis., 170:795-801 (1994); and Muñoz-Alia et al., VirusResearch, 236: 30-43 (2017)). This could complicate futureinterpretations since antigenic drift in Stealth was modeled bymonoclonal antibodies induced by the vaccine virus. CDV-vaccinatedferrets were used as a negative and positive control for neutralizationof vaccine and Stealth virus, respectively. Sera #126, #128, #129, #134,#136 and #137, used in FIG. 12 could not be tested due to materialscarcity. NT₅₀ values of the samples tested showed an overall geometricmean 5.39-fold lower (2.43 antigenic units) in neutralization potencyagainst Stealth virus, ranging from 3.12 to 10.9-fold (FIG. 14C). Serum152 yielded a less than 4-fold lower (1.64 antigenic units) ND₅₀ titeragainst Stealth virus and that of serum 131 and 157 were in thethreshold (4-fold). Of all sera tested, serum 152 was the only serashowing protective levels against Stealth virus (430 mIU/mL) and had thehighest NT₅₀ titer of all human sera tested against the vaccine strain(1344 mIU/mL). It was then tempting to speculate that the magnitude ofthe antibody response against the homotypic vaccine determined whetheror not protective levels against Stealth virus infection were achieved.Correlation analysis showed support of the hypothesis (Person r=0.9112;p<0.0001), suggesting a minimum NT₅₀ titer level of 926 mIU/mL forprotection against Stealth infection versus the current 210 mIU/mL useda predictor of seroconversion against the vaccine virus (Haralambieva etal., Vaccine, 29:4485-91 (2011)).

The following was performed to determine which extent the breadth andmagnitude of the antibody response impacted the antigenic variation seenin Stealth Virus. The antigenic variation was measured in a guinea pigmodel, which was shown to induce higher titers of cross-reactiveantibodies against influenza virus than of mice or ferrets (Nachbagaueret al., Nat. Immunol., 18(4):464-73 (2017)). In the context of MeV,guinea pigs mounted a highly neutralizing antibody response to thehomologous vaccine virus with an NT₅₀ value of 3584 mIU/mL (FIG. 14D).Conversely, the ND₅₀ titer against the heterologous the Stealth viruswas ˜6-fold lower (NT₅₀=563), and therefore antigenically significant.These results indicate that immunodominance was largely conserved acrossspecifies and that antigenic variation can be generated so as to escapethe protective levels induced upon vaccination.

Stealth is Solely CD46 Tropic

In influenza A viruses, receptor binding avidity and antigenic variationare closely related (Hensley et al., Science, 326:734-6 (2009); and Liet al., J. Virol., 87(17):9904-10 (2013)) and can compensate loss ofviral fitness (Kosik et al., PLoS Pathog., 14(1):e1006796 (2018)). Tobegin to address if receptor specificities are affected when nAb-escapemutations are present, CHO cells singly expressing the MeV receptorswere infected. Unexpectedly, given the intimately structural andfunctional interaction between CD46 and nectin-4 (FIG. 15), Stealthvirus proved to produce highly efficient CD46-dependent fusion but didnot show any in nectin-4-expressing CHO cells (FIG. 16A). Differences inreceptor density were excluded as potential explanation for thediscrimination of CD46 over nectin-4 usage since the number of moleculeson the cell surface was comparable between them two and it was 10-foldhigher than that on SLAM-expressing CHO cells (˜20,000) (FIG. 16B).Similar results were obtained when the panel of CHO cells were infectedwith Δ8 viruses, encoding the same MeV-HΔ8 as in Stealth, which arguesagainst allosteric interaction between MeV-H Δ8 and CDV-F as thecausative reason. However, further interactions with MeV matrix protein(MeV-M) could still influence receptor-binding interactions. Then,whether a transient transfection based fusion assay would parallel theresults observed in the context of the virus was studied. The approachwas to use MeV-F in combination with either MeV-H A or MeV-HΔ8. Theindicated that MeV-HΔ8 discriminated nectin-4 without significantlyaffected CD46-dependent fusion (FIG. 16C). Similar CD46-dependent fusionactivity was observed with both human and African green monkey cells(FIG. 17). The receptor binding disassociation constants were examinedfor the various recombinant MeV-H proteins (A, H1, and Δ8) by ELISA(FIG. 18). Unlike the anti-FLAG antibody used as a control, therecombinant cellular receptors showed differential binding avidity forthe MeV-H proteins (FIG. 16D). MeV-H Δ8 bound approximately 4000-foldbetter to CD46 than did MeV-H A and H1, with an apparent K_(d) of 190 pMversus 819 μM for MeV-H A (K_(d) was ambiguous). Binding values fornectin-4 were the worst across all three receptors, and saturation wasnot achieved at the highest concentration used. Even though nosignificant differences appeared for the apparent Ka of nectin-4 to therecombinant MeV-H proteins, B_(max) values for MeV-HΔ8 were decreasedapproximately by a half in comparison to MeV-H A and H1 (1.56, 1.14, and0.74, respectively). As expected, MeV-H Δ8 showed negligible binding toSLAM-Fc. MeV-H H1 showed lower binding to SLAM-Fc than MeV-H A, withK_(d) of 10.43 μM and 2.68 μM, but Bmax values were also lower (2.09 and1.19, respectively). Together, these results demonstrate that MeV-H Δ8discriminates CD46 usage over nectin-4 via an increase in the avidityinteraction with CD46 while decreasing that for nectin-4.

As described herein, 30 known antibody epitopes were systematicallyeliminated from the measles H glycoprotein. Viruses having that measlesH glycoprotein demonstrated resistance to neutralization by anti-Hantibodies present in measles-immune human, mouse and rabbit serum. Inaddition, substitution of the measles F glycoprotein with the homologousF protein of a related morbillivirus was used to generate MeV stealth, arecombinant MeV resistant to neutralization by measles-immune humanserum. The virus was shown to remain fully fusogenic and grow solely inCD46-positive cells with no cost in virus fitness. These resultsdemonstrate that the MeV stealth platform can be used for oncolyticvirotherapy in measles-immune cancer patients.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A recombinant virus comprising: a nucleic acid encoding a measles virus H polypeptide comprising at least six amino acid substitutions as compared to a wild-type measles virus H polypeptide; and a nucleic acid encoding a morbillivirus F polypeptide other than a measles virus F polypeptide.
 2. The virus of claim 1, wherein said virus is a measles virus.
 3. The virus of any one of claims 1-2, wherein said virus is an adenovirus.
 4. The virus of any one of claims 1-3, wherein said encoded measles virus H polypeptide and said morbillivirus F polypeptide are incorporated into the envelope of said recombinant virus.
 5. The virus of any one of claims 1-4, wherein said measles virus H polypeptide comprises SEQ ID NO:9 having six or more of the following amino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, and F552V.
 6. The virus of any one of claims 1-5, wherein said measles virus H polypeptide comprises SEQ ID NO:9 having the following amino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, and F552V.
 7. The virus of any one of claims 1-6, wherein said measles virus H polypeptide comprises at least one amino acid substitution within each of the antigenic sites set forth in Table
 1. 8. The virus of any one of claims 1-7, wherein said wild-type measles virus H polypeptide is a wild-type measles virus H polypeptide of the MVi/Madrid.SPA/50.10 strain.
 9. The virus of any one of claims 1-7, wherein said wild-type measles virus H polypeptide comprises the amino acid sequence set forth in SEQ ID NO:9.
 10. The virus of any one of claims 1-7, wherein said measles virus H polypeptide comprises SEQ ID NO:3.
 11. The virus of any one of claims 1-10, wherein said morbillivirus F polypeptide is a canine distemper virus F polypeptide.
 12. The virus of any one of claims 1-11, wherein said virus exhibits CD46-dependent cell entry.
 13. The virus of claim 12, wherein said virus exhibits reduced Nectin-4-dependent cell entry relative to a wild type virus.
 14. The virus of any one of claims 1-13, wherein said measles virus H polypeptide comprises at least one amino acid substitution within each of the antigenic sites set forth in Table
 2. 15. The virus of any one of claims 1-14, wherein said measles virus H polypeptide comprises a substitution at position E471 as compared to said wild-type measles virus H polypeptide.
 16. The virus of claim 15, wherein said substitution at position E471 is a E471K substitution.
 17. The virus of any one of claims 1-16, wherein said measles virus H polypeptide comprises SEQ ID NO:9 having the following amino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, E471K, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, and F552V.
 18. The virus of any one of claims 1-17, wherein said virus lacks said measles virus F polypeptide, lacks nucleic acid encoding said measles virus F polypeptide, or lacks both said measles virus F polypeptide and said nucleic acid encoding said measles virus F polypeptide.
 19. The virus of any one of claims 1-18, wherein said virus lacks said wild-type measles virus H polypeptide, lacks nucleic acid encoding said wild-type measles virus H polypeptide, or lacks both said wild-type measles virus H polypeptide and said nucleic acid encoding said wild-type measles virus H polypeptide.
 20. A method for reducing the number of viable tumor cells in a mammal, said method comprising administering to said mammal the virus of any one of claims 1-13.
 21. The method of claim 20, wherein said mammal is a human.
 22. A method for reducing the number of viable tumor cells in a mammal, said method comprising administering to said mammal the virus of any one of claims 14-19.
 23. The method of claim 22, wherein said mammal is a human.
 24. A method for stimulating an immune response against measles virus in a mammal, said method comprises administering to said mammal the measles virus of any one of claims 1-13.
 25. The method of claim 24, where said mammal is an infant.
 26. The method of claim 25, wherein said infant is a human infant.
 27. The method of claim 26, wherein said human infant has transplacentally acquired anti-measles antibodies.
 28. A method for stimulating an immune response against measles virus in a mammal, said method comprises administering to said mammal the measles virus of any one of claims 14-19.
 29. The method of claim 28, where said mammal is an infant.
 30. The method of claim 29, wherein said infant is a human infant.
 31. The method of claim 30, wherein said human infant has transplacentally acquired anti-measles antibodies.
 32. A nucleic acid construct comprising: a nucleic acid encoding a measles virus H polypeptide comprising at least six amino acid substitutions as compared to a wild-type measles virus H polypeptide, and a nucleic acid encoding a morbillivirus F polypeptide other than a measles virus F polypeptide.
 33. The nucleic acid construct of claim 32, wherein said measles virus H polypeptide comprises SEQ ID NO:9 having six or more of the following amino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, and F552V.
 34. The nucleic acid construct of claim 32, wherein said measles virus H polypeptide comprises SEQ ID NO:9 having the following amino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, N481Y, K488E, G491E, H495R, D505T, R533G S546G, R547G, and F552V.
 35. The nucleic acid construct of any one of claims 32-34, wherein said measles virus H polypeptide comprises at least one amino acid substitution within each of the antigenic sites set forth in Table
 1. 36. The nucleic acid construct of any one of claims 32-35, wherein said wild-type measles virus H polypeptide is a wild-type measles virus H polypeptide of the MVi/Madrid.SPA/50.10 strain.
 37. The nucleic acid construct of any one of claims 32-34, wherein said wild-type measles virus H polypeptide comprises the amino acid sequence set forth in SEQ ID NO:9.
 38. The nucleic acid construct of any one of claims 32-37, wherein said measles virus H polypeptide comprises SEQ ID NO:3.
 39. The nucleic acid construct of any one of claims 32-38, wherein said morbillivirus F polypeptide is a canine distemper virus F polypeptide.
 40. The nucleic acid construct of any one of claims 32-39, wherein said nucleic acid construct is a viral vector.
 41. The nucleic acid construct of any one of claims 32-40, wherein said viral vector is derived from a virus selected from the group consisting of an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a herpes virus, a vaccinia virus, and a rhabdovirus.
 42. The nucleic acid construct of any one of claims 32-41, wherein said measles virus H polypeptide comprises at least one amino acid substitution within each of the antigenic sites set forth in Table
 2. 43. The nucleic acid construct of any one of claims 32-42, wherein said measles virus H polypeptide comprises a substitution at position E471 as compared to said wild-type measles virus H polypeptide.
 44. The nucleic acid construct of claim 43, wherein said substitution at position E471 is a E471K substitution.
 45. The nucleic acid construct of any one of claims 32-44, wherein said measles virus H polypeptide comprises SEQ ID NO:9 having the following amino acid substitutions: S189P, E235G, N238D, L249P, G302R, Y310C, Q311R, R377Q, M378K, D416N, E471K, N481Y, K488E, G491E, H495R, D505T, R533G, S546G, R547G, and F552V.
 46. The nucleic acid construct of any one of claims 32-45, wherein said nucleic acid construct does not encode said measles virus F polypeptide.
 47. The nucleic acid construct of any one of claims 32-46, wherein said nucleic acid construct does not encode said wild-type measles virus H polypeptide. 